Modulation of Notch2 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of Notch2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding Notch2. Methods of using these compounds for modulation of Notch2 expression and for diagnosis and treatment of disease associated with expression of Notch2 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of Notch2. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingNotch2. Such compounds are shown herein to modulate the expression ofNotch2.

BACKGROUND OF THE INVENTION

[0002] Intrinsic, cell-autonomous factors as well as non-autonomous,short-range and long-range signals guide cells through distinctdevelopmental paths. An organism frequently uses the same signalingpathway within different cellular contexts to achieve uniquedevelopmental goals.

[0003] Notch signaling is an evolutionarily conserved mechanism used tocontrol cell fates through local cell interactions. The gene encodingthe original Notch receptor was discovered in Drosophila melanogasterdue to the fact that partial loss of function of the gene results innotches at the wing margin (Artavanis-Tsakonas et al., Science, 1999,284, 770-776). Signals transmitted through the Notch receptor, incombination with other cellular factors, influence differentiation,proliferation and apoptotic events at all stages of development(Artavanis-Tsakonas et al., Science, 1999, 284, 770-776).

[0004] Mature Notch proteins are heterodimeric receptors derived fromthe cleavage of Notch pre-proteins into an extracellular subunitcontaining multiple EGF-like repeats and a transmembrane subunitincluding the intracellular region (Blaumueller et al., Cell, 1997, 90,281-291). Notch activation results from the binding of ligands expressedby neighboring cells or soluble ligands and signaling from activatedNotch involves networks of transcription regulators (Artavanis-Tsakonaset al., Science, 1995, 268, 225-232).

[0005] In context of experimental cancer immunotherapy, the Notchsignaling network is acquiring increasing importance for its possibleroles in neoplastic cells and the immune system (Jang et al., Curr.Opin. Mol. Ther., 2000, 2, 55-65).

[0006] Four mammalian Notch homologs have been identified and aredesignated Notch1, Notch2, Notch3 and Notch4. Human Notch2 (also knownas Notch (Drosophila homolog 2) was identified and mapped to chromosome1p13-p11, a region associated with neoplasia-associated translocations(Larsson et al., Genomics, 1994, 24, 253-258). Larsson et al. predictedthat the human Notch genes are proto-oncogenes and candidates for sitesof chromosome breakage in neoplasia-associated translocations (Larssonet al., Genomics, 1994, 24, 253-258).

[0007] Disclosed and claimed in U.S. Pat. No. 5,789,195 are nucleic acidsequences encoding Notch genes. Antibodies to human Notch proteins areadditionally provided (Artavanis-Tsakonas et al., 1998). Amino acidsequences of Notch genes and antibodies against Notch proteins are alsodisclosed and claimed in U.S. Pat. No. 6,090,922 (Artavanis-Tsakonas etal., 2000).

[0008] Modulation of expression of Notch genes may prove to be a usefulpoint for therapeutic intervention in developmental, hyperproliferativeor autoimmune disorders or disorders arising from aberrant apoptosis.

[0009] Methods for producing allergen- or antigen-tolerant Tcellsemploying compositions capable of upregulating expression of anendogenous Notch protein are disclosed and claimed in PCT publication WO00/36089 (Lamb et al., 2000).

[0010] Disclosed and claimed in U.S. Pat. No. 6,149,902 is a method forcell transplantation which includes contacting a precursor cell with anagonist of Notch function effective to inhibit differentiation of thecell wherein said agonist is a Delta protein, a Serrate protein or anantibody to a Notch protein (Artavanis-Tsakonas et al., 2000).

[0011] Disclosed in U.S. Pat. No. 6,083,904 and PCT publication WO94/07474 are therapeutic and diagnostic methods and compositions basedon Notch proteins and nucleic acids, wherein antisense methods aregenerally disclosed (Artavanis-Tsakonas, 2000; Artavanis-Tsakonas etal., 1994).

[0012] Disclosed and claimed in U.S. Pat. No. 5,786,158 are methods andcompositions for the detection of malignancy or nervous system disordersbased on the level of Notch proteins or nucleic acids(Artavanis-Tsakonas et al., 1998).

[0013] Disclosed and claimed in PCT publication WO 00/20576 are methodsfor inducing differentiation and apoptosis in human cells that overexpress Notch proteins wherein Notch function is disrupted usingantisense oligonucleotides that target the EGF repeat region, the 1in/notch region and the ankyrin region (Miele et al., 2000).

[0014] Disclosed and claimed in PCT publication WO 01/25422 is anantisense oligonucleotide directed to exon 22 of human Notch2 (Bartelmezand Iversen, 2001).

[0015] Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of Notch2. To date, investigative strategies aimedat modulating Notch2 expression have involved the use of antibodies andNotch-regulating proteins as well as antisense RNA and oligonucleotides.Consequently, there remains a long felt need for additional agentscapable of effectively inhibiting Notch2 function.

[0016] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of Notch2.

[0017] The present invention provides compositions and methods formodulating expression of Notch2.

SUMMARY OF THE INVENTION

[0018] The present invention is directed to compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding Notch2, and which modulate the expression ofNotch2. Pharmaceutical and other compositions comprising the compoundsof the invention are also provided. Further provided are methods ofscreening for modulators of Notch2 and methods of modulating theexpression of Notch2 in cells, tissues or animals comprising contactingsaid cells, tissues or animals with one or more of the compounds orcompositions of the invention. Methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of Notch2 are also set forthherein. Such methods comprise administering a therapeutically orprophylactically effective amount of one or more of the compounds orcompositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0019] A. Overview of the Invention

[0020] The present invention employs compounds, preferablyoligonucleotides and similar species for use in modulating the functionor effect of nucleic acid molecules encoding Notch2. This isaccomplished by providing oligonucleotides which specifically hybridizewith one or more nucleic acid molecules encoding Notch2. As used herein,the terms “target nucleic acid” and “nucleic acid molecule encodingNotch2” have been used for convenience to encompass DNA encoding Notch2,RNA (including pre-mRNA and mRNA or portions thereof) transcribed fromsuch DNA, and also cDNA derived from such RNA. The hybridization of acompound of this invention with its target nucleic acid is generallyreferred to as “antisense”. Consequently, the preferred mechanismbelieved to be included in the practice of some preferred embodiments ofthe invention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently preferred to target specificnucleic acid molecules and their functions for such antisenseinhibition.

[0021] The functions of DNA to be interfered with can includereplication and transcription. Replication and transcription, forexample, can be from an endogenous cellular template, a vector, aplasmid construct or otherwise. The functions of RNA to be interferedwith can include functions such as translocation of the RNA to a site ofprotein translation, translocation of the RNA to sites within the cellwhich are distant from the site of RNA synthesis, translation of proteinfrom the RNA, splicing of the RNA to yield one or more RNA species, andcatalytic activity or complex formation involving the RNA which may beengaged in or facilitated by the RNA. One preferred result of suchinterference with target nucleic acid function is modulation of theexpression of Notch2. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

[0022] In the context of this invention, “hybridization” means thepairing of complementary strands of oligomeric compounds. In the presentinvention, the preferred mechanism of pairing involves hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases(nucleobases) of the strands of oligomeric compounds. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. Hybridization can occur under varyingcircumstances.

[0023] An antisense compound is specifically hybridizable when bindingof the compound to the target nucleic acid interferes with the normalfunction of the target nucleic acid to cause a loss of activity, andthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

[0024] In the present invention the phrase “stringent hybridizationconditions” or “stringent conditions” refers to conditions under which acompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances andin the context of this invention, “stringent conditions” under whicholigomeric compounds hybridize to a target sequence are determined bythe nature and composition of the oligomeric compounds and the assays inwhich they are being investigated.

[0025] “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleobases of an oligomeric compound. Forexample, if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

[0026] It is understood in the art that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the antisensecompounds of the present invention comprise at least 70% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise 90% sequence complementarity and even morepreferably comprise 95% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

[0027] B. Compounds of the Invention

[0028] According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid. One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

[0029] While the preferred form of antisense compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

[0030] The first evidence that dsRNA could lead to gene silencing inanimals came in 1995 from work in the nematode, Caenorhabditis elegans(Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. haveshown that the primary interference effects of dsRNA areposttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

[0031] In the context of this invention, the term “oligomeric compound”refers to a polymer or oligomer comprising a plurality of monomericunits. In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologsthereof. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for a target nucleic acid and increased stability inthe presence of nucleases.

[0032] While oligonucleotides are a preferred form of the compounds ofthis invention, the present invention comprehends other families ofcompounds as well, including but not limited to oligonucleotide analogsand mimetics such as those described herein.

[0033] The compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength.

[0034] In one preferred embodiment, the compounds of the invention are12 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length.

[0035] In another preferred embodiment, the compounds of the inventionare 15 to 30 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0036] Particularly preferred compounds are oligonucleotides from about12 to about 50 nucleobases, even more preferably those comprising fromabout 15 to about 30 nucleobases.

[0037] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0038] Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

[0039] C. Targets of the Invention

[0040] “Targeting” an antisense compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose function is to be modulated. This target nucleic acid may be,for example, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes Notch2.

[0041] The targeting process usually also includes determination of atleast one target region, segment, or site within the target nucleic acidfor the antisense interaction to occur such that the desired effect,e.g., modulation of expression, will result. Within the context of thepresent invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable structure,function, or characteristic. Within regions of target nucleic acids aresegments. “Segments” are defined as smaller or sub-portions of regionswithin a target nucleic acid. “Sites,” as used in the present invention,are defined as positions within a target nucleic acid.

[0042] Since, as is known in the art, the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding Notch2, regardless of the sequence(s)of such codons. It is also known in the art that a translationtermination codon (or “stop codon”) of a gene may have one of threesequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNAsequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0043] The terms “start codon region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation initiation codon. Similarly, the terms“stop codon region” and “translation termination codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation termination codon. Consequently, the “start codonregion” (or “translation initiation codon region”) and the “stop codonregion” (or “translation termination codon region”) are all regionswhich may be targeted effectively with the antisense compounds of thepresent invention.

[0044] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

[0045] Other target regions include the 5′ untranslated region (5′UTR),known in the art to refer to the portion of an mRNA in the 5′ directionfrom the translation initiation codon, and thus including nucleotidesbetween the 5′ cap site and the translation initiation codon of an mRNA(or corresponding nucleotides on the gene), and the 3′ untranslatedregion (3′UTR), known in the art to refer to the portion of an mRNA inthe 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA (or corresponding nucleotides on the gene). The 5′ capsite of an mRNA comprises an N7-methylated guanosine residue joined tothe 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′cap region of an mRNA is considered to include the 5′ cap structureitself as well as the first 50 nucleotides adjacent to the cap site. Itis also preferred to target the 5′ cap region.

[0046] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. Targeting splice sites,i.e., intron-exon junctions or exon-intron junctions, may also beparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred target sites. mRNA transcripts producedvia the process of splicing of two (or more) mRNAs from different genesources are known as “fusion transcripts”. It is also known that intronscan be effectively targeted using antisense compounds targeted to, forexample, DNA or pre-mRNA.

[0047] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

[0048] Upon excision of one or more exon or intron regions, or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0049] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

[0050] The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

[0051] While the specific sequences of certain preferred target segmentsare set forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

[0052] Target segments 8-80 nucleobases in length comprising a stretchof at least eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

[0053] Target segments can include DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

[0054] Once one or more target regions, segments or sites have beenidentified, antisense compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

[0055] D. Screening and Target Validation

[0056] In a further embodiment, the “preferred target segments”identified herein may be employed in a screen for additional compoundsthat modulate the expression of Notch2. “Modulators” are those compoundsthat decrease or increase the expression of a nucleic acid moleculeencoding Notch2 and which comprise at least an 8-nucleobase portionwhich is complementary to a preferred target segment. The screeningmethod comprises the steps of contacting a preferred target segment of anucleic acid molecule encoding Notch2 with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingNotch2. Once it is shown that the candidate modulator or modulators arecapable of modulating (e.g. either decreasing or increasing) theexpression of a nucleic acid molecule encoding Notch2, the modulator maythen be employed in further investigative studies of the function ofNotch2, or for use as a research, diagnostic, or therapeutic agent inaccordance with the present invention.

[0057] The preferred target segments of the present invention may bealso be combined with their respective complementary antisense compoundsof the present invention to form stabilized double-stranded (duplexed)oligonucleotides.

[0058] Such double stranded oligonucleotide moieties have been shown inthe art to modulate target expression and regulate translation as wellas RNA processsing via an antisense mechanism. Moreover, thedouble-stranded moieties may be subject to chemical modifications (Fireet al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395,854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science,1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998,95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197;Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev.2001, 15, 188-200). For example, such double-stranded moieties have beenshown to inhibit the target by the classical hybridization of antisensestrand of the duplex to the target, thereby triggering enzymaticdegradation of the target (Tijsterman et al., Science, 2002, 295,694-697).

[0059] The compounds of the present invention can also be applied in theareas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between Notch2 and a disease state, phenotype, or condition.These methods include detecting or modulating Notch2 comprisingcontacting a sample, tissue, cell, or organism with the compounds of thepresent invention, measuring the nucleic acid or protein level of Notch2and/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further compound of the invention. Thesemethods can also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

[0060] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0061] The compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

[0062] For use in kits and diagnostics, the compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues.

[0063] As one nonlimiting example, expression patterns within cells ortissues treated with one or more antisense compounds are compared tocontrol cells or tissues not treated with antisense compounds and thepatterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds which affect expression patterns.

[0064] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0065] The compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingNotch2. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective Notch2 inhibitors will also be effective primers or probesunder conditions favoring gene amplification or detection, respectively.These primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding Notch2 and in theamplification of said nucleic acid molecules for detection or for use infurther studies of Notch2. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding Notch2 can be detected by means known inthe art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of Notch2 in a sample may also be prepared.

[0066] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisense compounds havebeen employed as therapeutic moieties in the treatment of disease statesin animals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

[0067] For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Notch2 is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of a Notch2inhibitor. The Notch2 inhibitors of the present invention effectivelyinhibit the activity of the Notch2 protein or inhibit the expression ofthe Notch2 protein. In one embodiment, the activity or expression ofNotch2 in an animal is inhibited by about 10%. Preferably, the activityor expression of Notch2 in an animal is inhibited by about 30%. Morepreferably, the activity or expression of Notch2 in an animal isinhibited by 50% or more.

[0068] For example, the reduction of the expression of Notch2 may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within saidfluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding Notch2 protein and/or the Notch2 protein itself.

[0069] The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

[0070] F. Modifications

[0071] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0072] Modified Internucleoside Linkages (Backbones)

[0073] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0074] Preferred modified oligonucleotide backbones containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. asingle-inverted nucleoside residue which may be abasic (the nucleobaseis missing or has a hydroxyl group in place thereof). Various salts,mixed salts and free acid forms are also included.

[0075] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0076] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0077] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0078] Modified Sugar and Internucleoside Linkages-Mimetics

[0079] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage (i.e. the backbone), of the nucleotide unitsare replaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0080] Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0081] Modified Sugars

[0082] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃. SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy(also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0083] Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl(2′-CH₂—CH═CH₂),2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0084] A further preferred modification of the sugar includes LockedNucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugarmoiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0085] Natural and Modified Nucleobases

[0086] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivativesof pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2-H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

[0087] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0088] Conjugates

[0089] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. These moieties or conjugates caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of whichare incorporated herein by reference. Conjugate moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

[0090] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0091] Chimeric Compounds

[0092] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

[0093] The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0094] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0095] G. Formulations

[0096] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0097] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0098] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0099] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligonucleotides, preferred examplesof pharmaceutically acceptable salts and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

[0100] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0101] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0102] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0103] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

[0104] Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

[0105] Formulations of the present invention include liposomalformulations. As used in the present invention, the term “liposome”means a vesicle composed of amphiphilic lipids arranged in a sphericalbilayer or bilayers. Liposomes are unilamellar or multilamellar vesicleswhich have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes which are believed tointeract with negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

[0106] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0107] The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0108] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

[0109] One of skill in the art will recognize that formulations areroutinely designed according to their intended use, i.e. route ofadministration.

[0110] Preferred formulations for topical administration include thosein which the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA).

[0111] For topical or other administration, oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters, pharmaceutically acceptablesalts thereof, and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999, which is incorporated herein byreference in its entirety.

[0112] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts and fatty acids and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety. Also preferred are combinations of penetration enhancers,for example, fatty acids/salts in combination with bile acids/salts. Aparticularly preferred combination is the sodium salt of lauric acid,capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety. Oral formulations for oligonucleotides and theirpreparation are described in detail in U.S. application Ser. Nos.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

[0113] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0114] Certain embodiments of the invention provide pharmaceuticalcompositions containing one or more oligomeric compounds and one or moreother chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to cancer chemotherapeutic drugs such as daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0115] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Alternatively,compositions of the invention may contain two or more antisensecompounds targeted to different regions of the same nucleic acid target.Numerous examples of antisense compounds are known in the art. Two ormore combined compounds may be used together or sequentially.

[0116] H. Dosing

[0117] The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0118] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0119] Synthesis of Nucleoside Phosphoramidites

[0120] The following compounds, including amidites and theirintermediates were prepared as described in U.S. Pat. No. 6,426,220 andpublished PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediatefor 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineintermediate for 5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-⁴-benzoyl-5-methylcytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4-′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0121] Oligonucleotide and Oligonucleoside Synthesis

[0122] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0123] Oligonucleotides: Unsubstituted and substituted phosphodiester(P═O) oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0124] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0125] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0126] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. No. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0127] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0128] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0129] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0130] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0131] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

[0132] Oligonucleosides: Methylenemethylimino linked oligonucleosides,also identified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0133] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0134] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0135] RNA Synthesis

[0136] In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

[0137] Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

[0138] RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

[0139] Following synthesis, the methyl protecting groups on thephosphates are cleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

[0140] The 2′-orthoester groups are the last protecting groups to beremoved. The ethylene glycol monoacetate orthoester protecting groupdeveloped by Dharmacon Research, Inc. (Lafayette, Colo.), is one exampleof a useful orthoester protecting group which, has the followingimportant properties. It is stable to the conditions of nucleosidephosphoramidite synthesis and oligonucleotide synthesis. However, afteroligonucleotide synthesis the oligonucleotide is treated withmethylamine which not only cleaves the oligonucleotide from the solidsupport but also removes the acetyl groups from the orthoesters. Theresulting 2-ethyl-hydroxyl substituents on the orthoester are lesselectron withdrawing than the acetylated precursor. As a result, themodified orthoester becomes more labile to acid-catalyzed hydrolysis.Specifically, the rate of cleavage is approximately 10 times fasterafter the acetyl groups are removed. Therefore, this orthoesterpossesses sufficient stability in order to be compatible witholigonucleotide synthesis and yet, when subsequently modified, permitsdeprotection to be carried out under relatively mild aqueous conditionscompatible with the final RNA oligonucleotide product.

[0141] Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

[0142] RNA antisense compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense compounds can then be annealed by methodsknown in the art to form double stranded (duplexed) antisense compounds.For example, duplexes can be formed by combining 30 μl of each of thecomplementary strands of RNA oligonucleotides (50 uM RNA oligonucleotidesolution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisensecompounds can be used in kits, assays, screens, or other methods toinvestigate the role of a target nucleic acid.

Example 4

[0143] Synthesis of Chimeric Oligonucleotides

[0144] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0145] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0146] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0147] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[0148] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0149] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0150] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

[0151] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0152] Design and Screening of Duplexed Antisense Compounds TargetingNotch2

[0153] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target Notch2. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

[0154] For example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

[0155] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 uM. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5× solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0156] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate Notch2 expression.

[0157] When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6

[0158] Oligonucleotide Isolation

[0159] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0160] Oligonucleotide Synthesis—96 Well Plate Format

[0161] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0162] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0163] Oligonucleotide Analysis —96-Well Plate Format

[0164] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0165] Cell Culture and Oligonucleotide Treatment

[0166] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0167] T-24 Cells:

[0168] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

[0169] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0170] A549 Cells:

[0171] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0172] NHDF Cells:

[0173] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0174] HEK Cells:

[0175] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0176] Treatment with Antisense Compounds:

[0177] When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0178] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10

[0179] Analysis of Oligonucleotide Inhibition of Notch2 Expression

[0180] Antisense modulation of Notch2 expression can be assayed in avariety of ways known in the art. For example, Notch2 mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are well known in theart. Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

[0181] Protein levels of Notch2 can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toNotch2 can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 11

[0182] Design of Phenotypic Assays and In Vivo Studies for the Use ofNotch2 Inhibitors

[0183] Phenotypic Assays

[0184] Once Notch2 inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of Notch2 in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

[0185] In one non-limiting example, cells determined to be appropriatefor a particular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with Notch2inhibitors identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

[0186] Phenotypic endpoints include changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, lipids, nucleic acids, hormones, saccharides ormetals. Measurements of cellular status which include pH, stage of thecell cycle, intake or excretion of biological indicators by the cell,are also endpoints of interest.

[0187] Analysis of the geneotype of the cell (measurement of theexpression of one or more of the genes of the cell) after treatment isalso used as an indicator of the efficacy or potency of the Notch2inhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

[0188] In Vivo Studies

[0189] The individual subjects of the in vivo studies described hereinare warm-blooded vertebrate animals, which includes humans.

[0190] The clinical trial is subjected to rigorous controls to ensurethat individuals are not unnecessarily put at risk and that they arefully informed about their role in the study. To account for thepsychological effects of receiving treatments, volunteers are randomlygiven placebo or Notch2 inhibitor. Furthermore, to prevent the doctorsfrom being biased in treatments, they are not informed as to whether themedication they are administering is a Notch2 inhibitor or a placebo.Using this randomization approach, each volunteer has the same chance ofbeing given either the new treatment or the placebo.

[0191] Volunteers receive either the Notch2 inhibitor or placebo foreight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingNotch2 or Notch2 protein levels in body fluids, tissues or organscompared to pretreatment levels. Other measurements include, but are notlimited to, indices of the disease state or condition being treated,body weight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

[0192] Information recorded for each patient includes age (years),gender, height (cm), family history of disease state or condition(yes/no), motivation rating (some/moderate/great) and number and type ofprevious treatment regimens for the indicated disease or condition.

[0193] Volunteers taking part in this study are healthy adults (age 18to 65 years) and roughly an equal number of males and femalesparticipate in the study. Volunteers with certain characteristics areequally distributed for placebo and Notch2 inhibitor treatment. Ingeneral, the volunteers treated with placebo have little or no responseto treatment, whereas the volunteers treated with the Notch2 inhibitorshow positive trends in their disease state or condition index at theconclusion of the study.

Example 12

[0194] RNA Isolation

[0195] Poly(A)+ mRNA Isolation

[0196] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare routine in the art. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA,0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was addedto each well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

[0197] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

[0198] Total RNA Isolation

[0199] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 140 μL of RNAse free water into eachwell, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0200] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0201] Real-Time Quantitative PCR Analysis of Notch2 mRNA Levels

[0202] Quantitation of Notch2 mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

[0203] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0204] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofDATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0205] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0206] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

[0207] Probes and primers to human Notch2 were designed to hybridize toa human Notch2 sequence, using published sequence information (a genomicsequence of human Notch2 is represented by the complement of residues4894439-5015872 of GenBank accession number NT_(—)004754.7, incorporatedherein as SEQ ID NO: 4). For human Notch2 the PCR primers were: forwardprimer: TGGCAACTAACGTAGAAACTCAACA (SEQ ID NO: 5) reverse primer:TGCCAAGAGCATGAATACAGAGA (SEQ ID NO: 6) and the PCR probe was:FAM-ACAACTATAGACTTGCTCATTGTTCAGACTGATTGCC-TAMRA

[0208] (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were: forward primer:GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC(SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRAis the quencher dye.

Example 14

[0209] Northern Blot Analysis of Notch2 mRNA Levels

[0210] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0211] To detect human Notch2, a human Notch2 specific probe wasprepared by PCR using the forward primer TGGCAACTAACGTAGAAACTCAACA (SEQID NO: 5) and the reverse primer TGCCAAGAGCATGAATACAGAGA (SEQ ID NO: 6).To normalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0212] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0213] Antisense Inhibition of Human Notch2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0214] In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the human Notch2RNA, using published sequences (a genomic sequence of human Notch2represented by the complement of residues 4894439-5015872 of GenBankaccession number NT_(—)004754.7, incorporated herein as SEQ ID NO: 4;GenBank accession number NM_(—)024408.1, incorporated herein as SEQ IDNO: 11; GenBank accession number BC010154.1 incorporated herein as SEQID NO: 12; and GenBank accession number BI562298.1, incorporated hereinas SEQ ID NO: 13). The compounds are shown in Table 1. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds. All compounds in Table 1 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl(2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanNotch2 mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments in which A549cells were treated with the oligonucleotides of the present invention.The positive control for each datapoint is identified in the table bysequence ID number. If present, “N.D.” indicates “no data”. TABLE 1Inhibition of human Notch2 mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQID TARGET % SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO NO226896 Coding 4 25923 Tctcgatgttgacaatattc 58 14 1 226897 Coding 11 418Cactccttacctgtaaaccc 24 15 1 226898 Coding 4 34233 Ggcatttgcaggagaactgg67 16 1 226899 Coding 4 44403 Gtgctcccttcaaaacctgg 62 17 1 226900 Coding4 63879 Tttcctgcatgctcacaagg 79 18 1 226901 Coding 4 63923Ccttcagacactcacagtgg 76 19 1 226902 Coding 11 1455 Ctttgaaacctggcatgcac56 20 1 226903 Coding 4 64999 Caatgcacacctttgaaacc 32 21 1 226904 Coding4 65997 Tagccattcgggtgatcgat 73 22 1 226905 Coding 4 66002attcatagccattcgggtga 49 23 1 226906 Coding 4 66007 ctggcattcatagccattcg61 24 1 226907 Coding 4 66013 ggcacactggcattcatagc 41 25 1 226908 Coding4 66018 cctgtggcacactggcattc 53 26 1 226909 Coding 4 67768ttgcagatgcaggtgtagga 69 27 1 226910 Coding 4 67813 cattcatcaatctggtcact44 28 1 226911 Coding 4 71996 gtcatcaaaattaatttcac 50 29 1 226912 Coding4 72001 gcacagtcatcaaaattaat 66 30 1 226913 Coding 4 76276tggaggcacactcatcaatg 71 31 1 226914 Coding 4 76376 ttcacctgtgagtagcagct76 32 1 226915 Coding 4 76424 agacctccagtacagtttcc 53 33 1 226916 Coding4 76429 cactgagacctccagtacag 68 34 1 226917 Coding 4 80699aagcaggttccttggttcag 72 35 1 226918 Coding 4 82461 tgccagccaggagcacacaa68 36 1 226919 Coding 11 2755 tggcaaggattggcaaggca 73 37 1 226920 Coding4 89815 ttcatgtictgtctggcactt 74 38 1 226921 Coding 11 3181agggtctgacagtttttccc 62 39 1 226922 Coding 11 3186 tcaccagggtctgacagttt65 40 1 226923 Coding 4 94030 gctggcacaagtgttcaaca 69 41 1 226924 Coding4 95897 tcacagttgacaccctgata 80 42 1 226925 Coding 4 95902catactcacagttgacaccc 30 43 1 226926 Coding 4 95907 cacttcatactcacagttga59 44 1 226927 Coding 4 102378 tccccagcaaagccaggcaa 75 45 1 226928Coding 4 105700 acttgtccacagctgctctg 68 46 1 226929 Coding 4 106009ccatcccactggcaggcatg 76 47 1 226930 Coding 4 106099 tcatcacactggttgttgat77 48 1 226931 Coding 4 106135 aagttgtcaaacaggcactc 56 49 1 226932Coding 11 4519 cagtatttgtcatacttgca 64 50 1 226933 Coding 4 107505ctgcacagtatttgtcatac 80 51 1 226934 Coding 4 107510 gtggtctgcacagtatttgt66 52 1 226935 Coding 4 107515 ttgaagtggtctgcacagta 69 53 1 226936Coding 4 107520 tgtctttgaagtggtctgca 76 54 1 226937 Coding 4 107530tcacagtggttgtctttgaa 61 55 1 226938 Coding 11 4862 gactttagagccagccacct41 56 1 226939 Coding 4 111150 agcttcaaggtgctgctgtg 59 57 1 226940Coding 4 111155 tctgcagcttcaaggtgctg 56 58 1 226941 Coding 4 111897gcctcctcggagagaagcca 70 59 1 226942 Coding 4 111902 gagctgcctcctcggagaga45 60 1 226943 Coding 4 111965 accaagtctgtgatgatgtt 68 61 1 226944Coding 4 111970 ggtagaccaagtctgtgatg 44 62 1 226945 Coding 4 112028gcaaggtgcagggccatctc 44 63 1 226946 Coding 11 5926 tttccatggtcatccactgc74 64 1 226947 Coding 11 5931 cagattttccatggtcatcc 72 65 1 226948 Coding4 113747 acattattgacagcagctgc 66 66 1 226949 Coding 4 115116gcctccttggcaaggttagg 69 67 1 226950 Coding 4 115805 aagccacactgggcaaacgg64 68 1 226951 Coding 4 115854 atggtctgagctacctgccc 69 69 1 226952Coding 4 115890 cccacagaggctgggaaagg 78 70 1 226953 Stop 4 115895acttgcccacagaggctggg 60 71 1 Codon 226954 3′UTR 4 116067gtcacatctgaccagtcaga 71 72 1 226955 3′UTR 4 116072 tggtggtcacatctgaccag59 73 1 226956 3′UTR 4 116172 ggactctctcacgcataaac 68 74 1 226957 3′UTR4 116695 aaaggtcttgccctataaca 44 75 1 226958 3′UTR 4 117121cattccaaacctttttctgg 64 76 1 226959 Intron 4 118110 cagacaaatcaggtaagtgg75 77 1 226960 Intron: 4 119090 caaaacattacacctttggt 65 78 1 exonjunction 226961 Intron 4 119806 atttcacttaaggaatgtta 45 79 1 226962Intron: 4 19667 tcaggagatcgagaccatcc 41 80 1 exon junction 226963Intron: 4 34483 agctccttacctggaaggca 61 81 1 exon junction 226964Intron: 4 47121 ccaaccactacgggtcttgg 50 82 1 exon junction 226965Intron: 4 67672 ccagtgaaacctttggaaag 53 83 1 exon junction 226966 Intron4 82473 atgttcttaccttgccagcc 29 84 1 226967 5′UTR 4 83067ggcatactcactggcaaggc 60 85 1 226968 5′UTR 4 106175 aagcccttacttgcatgtct80 86 1 226969 5′UTR 4 108223 gaatgacagagcaactgaag 57 87 1 226970 5′UTR12 8 ctttctcgggtgtgcagccc 16 88 1 226971 5′UTR 12 239gtatcttctcggtcgcctcc 55 89 1 226972 5′UTR 12 1027 cctgtctctttcccagagct37 90 1 226973 5′UTR 13 29 cgccgccttgggcacccagg 16 91 1

[0215] As shown in Table 1, SEQ ID NOs 14, 16, 17, 18, 19, 20, 22, 24,26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59, 61, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 81, 82, 83, 85, 86, 87 and 89demonstrated at least 50% inhibition of human Notch2 expression in thisassay and are therefore preferred. More preferred are SEQ ID Nos: 42,51, and 86. The target regions to which these preferred sequences arecomplementary are herein referred to as “preferred target segments” andare therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 2. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 2 is the species in whicheach of the preferred target segments was found. TABLE 2 Sequence andposition of preferred target segments identified in Notch2. TARGET SEQID TARGET REV COMP SEQ ID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO143548 4 25923 gaatattgtcaacatcgaga 14 H. sapiens 92 143550 4 34233ccagttctcctgcaaatgcc 16 H. sapiens 93 143551 4 44403ccaggttttgaagggagcac 17 H. sapiens 94 143552 4 63879ccttgtgagcatgcaggaaa 18 H. sapiens 95 143553 4 63923ccactgtgagtgtctgaagg 19 H. sapiens 96 143554 11 1455gtgcatgccaggtttcaaag 20 H. sapiens 97 143556 4 65997atcgatcacccgaatggcta 22 H. sapiens 98 143558 4 66007cgaatggctatgaatgccag 24 H. sapiens 99 143560 4 66018gaatgccagtgtgccacagg 26 H. sapiens 100 143561 4 67768tcctacacctgcatctgcaa 27 H. sapiens 101 143563 4 71996gtgaaattaattttgatgac 29 H. sapiens 102 143564 4 72001attaattttgatgactgtgc 30 H. sapiens 103 143565 4 76276cattgatgagtgtgcctcca 31 H. sapiens 104 143566 4 76376agctgctactcacaggtgaa 32 H. sapiens 105 143567 4 76424ggaaactgtactggaggtct 33 H. sapiens 106 143568 4 76429ctgtactggaggtctcagtg 34 H. sapiens 107 143569 4 80699ctgaaccaaggaacctgctt 35 H. sapiens 108 143570 4 82461ttgtgtgctcctggctggca 36 H. sapiens 109 143571 11 2755tgccttgccaatccttgcca 37 H. sapiens 110 143572 4 89815aagtgccagacagacatgaa 38 H. sapiens 111 143573 11 3181gggaaaaactgtcagaccct 39 H. sapiens 112 143574 11 3186aaactgtcagaccctggtga 40 H. sapiens 113 143575 4 94030tgttgaacacttgtgccagc 41 H. sapiens 114 143576 4 95897tatcagggtgtcaactgtga 42 H. sapiens 115 143578 4 95907tcaactgtgagtatgaagtg 44 H. sapiens 116 143579 4 102378ttgcctggctttgctgggga 45 H. sapiens 117 143580 4 105700cagagcagctgtggacaagt 46 H. sapiens 118 143581 4 106009catgcctgccagtgggatgg 47 H. sapiens 119 143582 4 106099atcaacaaccagtgtgatga 48 H. sapiens 120 143583 4 106135gagtgcctgtttgacaactt 49 H. sapiens 121 143584 11 4519tgcaagtatgacaaatactg 50 H. sapiens 122 143585 4 107505gtatgacaaatactgtgcag 51 H. sapiens 123 143586 4 107510acaaatactgtgcagaccac 52 H. sapiens 124 143587 4 107515tactgtgcagaccacttcaa 53 H. sapiens 125 143588 4 107520tgcagaccacttcaaagaca 54 H. sapiens 126 143589 4 107530ttcaaagacaaccactgtga 55 H. sapiens 127 143591 4 111150cacagcagcaccttgaagct 57 H. sapiens 128 143592 4 111155cagcaccttgaagctgcaga 58 H. sapiens 129 143593 4 111897tggcttctctccgaggaggc 59 H. sapiens 130 143595 4 111965aacatcatcacagacttggt 61 H. sapiens 131 143598 11 5926gcagtggatgaccatggaaa 64 H. sapiens 132 143599 11 5931ggatgaccatggaaaatctg 65 H. sapiens 133 143600 4 113747gcagctgctgtcaataatgt 66 H. sapiens 134 143601 4 115116cctaaccttgccaaggaggc 67 H. sapiens 135 143602 4 115805ccgtttgcccagtgtggctt 68 H. sapiens 136 143603 4 115854gggcaggtagctcagaccat 69 H. sapiens 137 143604 4 115890cctttcccagcctctgtggg 70 H. sapiens 138 143605 4 115895cccagcctctgtgggcaagt 71 H. sapiens 139 143606 4 116067tctgactggtcagatgtgac 72 H. sapiens 140 143607 4 116072ctggtcagatgtgaccacca 73 H. sapiens 141 143608 4 116172gtttatgcgtgagagagtcc 74 H. sapiens 142 143610 4 117121ccagaaaaaggtttggaatg 76 H. sapiens 143 143611 4 118110ccacttacctgatttgtctg 77 H. sapiens 144 143612 4 119090accaaaggtgtaatgttttg 78 H. sapiens 145 143615 4 34483tgccttccaggtaaggagct 81 H. sapiens 146 143616 4 47121ccaagacccqtagtggttgg 82 H. sapiens 147 143617 4 67672ctttccaaaggtttcactgg 83 H. sapiens 148 143619 4 83067gccttgccagtgagtatgcc 85 H. sapiens 149 143620 4 106175agacatgcaagtaagggctt 86 H. sapiens 150 143621 4 108223cttcagttgctctgtcattc 87 H. sapiens 151 143623 12 239ggaggcgaccgagaagatac 89 H. sapiens 152

[0216] As these “preferred target segments” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of Notch2.

[0217] According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 16

[0218] Western Blot Analysis of Notch2 Protein Levels

[0219] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Notch2 is used, witha radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 152 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 11189 DNA Homosapiens CDS (13)...(7428) 4 gcgaccgaga ag atg ccc gcc ctg cgc ccc gctctg ctg tgg gcg ctg ctg 51 Met Pro Ala Leu Arg Pro Ala Leu Leu Trp AlaLeu Leu 1 5 10 gcg ctc tgg ctg tgc tgc gcg acc ccc gcg cat gca ttg cagtgt cga 99 Ala Leu Trp Leu Cys Cys Ala Thr Pro Ala His Ala Leu Gln CysArg 15 20 25 gat ggc tat gaa ccc tgt gta aat gaa gga atg tgt gtt acc taccac 147 Asp Gly Tyr Glu Pro Cys Val Asn Glu Gly Met Cys Val Thr Tyr His30 35 40 45 aat ggc aca gga tac tgc aaa tgt cca gaa ggc ttc ttg ggg gaatat 195 Asn Gly Thr Gly Tyr Cys Lys Cys Pro Glu Gly Phe Leu Gly Glu Tyr50 55 60 tgt caa cat cga gac ccc tgt gag aag aac cgc tgc cag aat ggt ggg243 Cys Gln His Arg Asp Pro Cys Glu Lys Asn Arg Cys Gln Asn Gly Gly 6570 75 act tgt gtg gcc cag gcc atg ctg ggg aaa gcc acg tgc cga tgt gcc291 Thr Cys Val Ala Gln Ala Met Leu Gly Lys Ala Thr Cys Arg Cys Ala 8085 90 tca ggg ttt aca gga gag gac tgc cag tac tcg aca tct cat cca tgc339 Ser Gly Phe Thr Gly Glu Asp Cys Gln Tyr Ser Thr Ser His Pro Cys 95100 105 ttt gtg tct cga ccc tgc ctg aat ggc ggc aca tgc cat atg ctc agc387 Phe Val Ser Arg Pro Cys Leu Asn Gly Gly Thr Cys His Met Leu Ser 110115 120 125 cgg gat acc tat gag tgc acc tgt caa gtc ggg ttt aca ggt aaggag 435 Arg Asp Thr Tyr Glu Cys Thr Cys Gln Val Gly Phe Thr Gly Lys Glu130 135 140 tgc caa tgg acc gat gcc tgc ctg tct cat ccc tgt gca aat ggaagt 483 Cys Gln Trp Thr Asp Ala Cys Leu Ser His Pro Cys Ala Asn Gly Ser145 150 155 acc tgt acc act gtg gcc aac cag ttc tcc tgc aaa tgc ctc acaggc 531 Thr Cys Thr Thr Val Ala Asn Gln Phe Ser Cys Lys Cys Leu Thr Gly160 165 170 ttc aca ggg cag aaa tgt gag act gat gtc aat gag tgt gac attcca 579 Phe Thr Gly Gln Lys Cys Glu Thr Asp Val Asn Glu Cys Asp Ile Pro175 180 185 gga cac tgc cag cat ggt ggc acc tgc ctc aac ctg cct ggt tcctac 627 Gly His Cys Gln His Gly Gly Thr Cys Leu Asn Leu Pro Gly Ser Tyr190 195 200 205 cag tgc cag tgc ctt cag ggc ttc aca ggc cag tac tgt gacagc ctg 675 Gln Cys Gln Cys Leu Gln Gly Phe Thr Gly Gln Tyr Cys Asp SerLeu 210 215 220 tat gtg ccc tgt gca ccc tcg cct tgt gtc aat gga ggc acctgt cgg 723 Tyr Val Pro Cys Ala Pro Ser Pro Cys Val Asn Gly Gly Thr CysArg 225 230 235 cag act ggt gac ttc act ttt gag tgc aac tgc ctt cca ggtttt gaa 771 Gln Thr Gly Asp Phe Thr Phe Glu Cys Asn Cys Leu Pro Gly PheGlu 240 245 250 ggg agc acc tgt gag agg aat att gat gac tgc cct aac cacagg tgt 819 Gly Ser Thr Cys Glu Arg Asn Ile Asp Asp Cys Pro Asn His ArgCys 255 260 265 cag aat gga ggg gtt tgt gtg gat ggg gtc aac act tac aactgc cgc 867 Gln Asn Gly Gly Val Cys Val Asp Gly Val Asn Thr Tyr Asn CysArg 270 275 280 285 tgt ccc cca caa tgg aca gga cag ttc tgc aca gag gatgtg gat gaa 915 Cys Pro Pro Gln Trp Thr Gly Gln Phe Cys Thr Glu Asp ValAsp Glu 290 295 300 tgc ctg ctg cag ccc aat gcc tgt caa aat ggg ggc acctgt gcc aac 963 Cys Leu Leu Gln Pro Asn Ala Cys Gln Asn Gly Gly Thr CysAla Asn 305 310 315 cgc aat gga ggc tat ggc tgt gta tgt gtc aac ggc tggagt gga gat 1011 Arg Asn Gly Gly Tyr Gly Cys Val Cys Val Asn Gly Trp SerGly Asp 320 325 330 gac tgc agt gag aac att gat gat tgt gcc ttc gcc tcctgt act cca 1059 Asp Cys Ser Glu Asn Ile Asp Asp Cys Ala Phe Ala Ser CysThr Pro 335 340 345 ggc tcc acc tgc atc gac cgt gtg gcc tcc ttc tct tgcatg tgc cca 1107 Gly Ser Thr Cys Ile Asp Arg Val Ala Ser Phe Ser Cys MetCys Pro 350 355 360 365 gag ggg aag gca ggt ctc ctg tgt cat ctg gat gatgca tgc atc agc 1155 Glu Gly Lys Ala Gly Leu Leu Cys His Leu Asp Asp AlaCys Ile Ser 370 375 380 aat cct tgc cac aag ggg gca ctg tgt gac acc aacccc cta aat ggg 1203 Asn Pro Cys His Lys Gly Ala Leu Cys Asp Thr Asn ProLeu Asn Gly 385 390 395 caa tat att tgc acc tgc cca caa ggc tac aaa ggggct gac tgc aca 1251 Gln Tyr Ile Cys Thr Cys Pro Gln Gly Tyr Lys Gly AlaAsp Cys Thr 400 405 410 gaa gat gtg gat gaa tgt gcc atg gcc aat agc aatcct tgt gag cat 1299 Glu Asp Val Asp Glu Cys Ala Met Ala Asn Ser Asn ProCys Glu His 415 420 425 gca gga aaa tgt gtg aac acg gat ggc gcc ttc cactgt gag tgt ctg 1347 Ala Gly Lys Cys Val Asn Thr Asp Gly Ala Phe His CysGlu Cys Leu 430 435 440 445 aag ggt tat gca gga cct cgt tgt gag atg gacatc aat gag tgc cat 1395 Lys Gly Tyr Ala Gly Pro Arg Cys Glu Met Asp IleAsn Glu Cys His 450 455 460 tca gac ccc tgc cag aat gat gct acc tgt ctggat aag att gga ggc 1443 Ser Asp Pro Cys Gln Asn Asp Ala Thr Cys Leu AspLys Ile Gly Gly 465 470 475 ttc aca tgt ctg tgc atg cca ggt ttc aaa ggtgtg cat tgt gaa tta 1491 Phe Thr Cys Leu Cys Met Pro Gly Phe Lys Gly ValHis Cys Glu Leu 480 485 490 gaa ata aat gaa tgt cag agc aac cct tgt gtgaac aat ggg cag tgt 1539 Glu Ile Asn Glu Cys Gln Ser Asn Pro Cys Val AsnAsn Gly Gln Cys 495 500 505 gtg gat aaa gtc aat cgt ttc cag tgc ctg tgtcct cct ggt ttc act 1587 Val Asp Lys Val Asn Arg Phe Gln Cys Leu Cys ProPro Gly Phe Thr 510 515 520 525 ggg cca gtt tgc cag att gat att gat gactgt tcc agt act ccg tgt 1635 Gly Pro Val Cys Gln Ile Asp Ile Asp Asp CysSer Ser Thr Pro Cys 530 535 540 ctg aat ggg gca aag tgt atc gat cac ccgaat ggc tat gaa tgc cag 1683 Leu Asn Gly Ala Lys Cys Ile Asp His Pro AsnGly Tyr Glu Cys Gln 545 550 555 tgt gcc aca ggt ttc act ggt gtg ttg tgtgag gag aac att gac aac 1731 Cys Ala Thr Gly Phe Thr Gly Val Leu Cys GluGlu Asn Ile Asp Asn 560 565 570 tgt gac ccc gat cct tgc cac cat ggt cagtgt cag gat ggt att gat 1779 Cys Asp Pro Asp Pro Cys His His Gly Gln CysGln Asp Gly Ile Asp 575 580 585 tcc tac acc tgc atc tgc aat ccc ggg tacatg ggc gcc atc tgc agt 1827 Ser Tyr Thr Cys Ile Cys Asn Pro Gly Tyr MetGly Ala Ile Cys Ser 590 595 600 605 gac cag att gat gaa tgt tac agc agccct tgc ctg aac gat ggt cgc 1875 Asp Gln Ile Asp Glu Cys Tyr Ser Ser ProCys Leu Asn Asp Gly Arg 610 615 620 tgc att gac ctg gtc aat ggc tac cagtgc aac tgc cag cca ggc acg 1923 Cys Ile Asp Leu Val Asn Gly Tyr Gln CysAsn Cys Gln Pro Gly Thr 625 630 635 tca ggg gtt aat tgt gaa att aat tttgat gac tgt gca agt aac cct 1971 Ser Gly Val Asn Cys Glu Ile Asn Phe AspAsp Cys Ala Ser Asn Pro 640 645 650 tgt atc cat gga atc tgt atg gat ggcatt aat cgc tac agt tgt gtc 2019 Cys Ile His Gly Ile Cys Met Asp Gly IleAsn Arg Tyr Ser Cys Val 655 660 665 tgc tca cca gga ttc aca ggg cag agatgt aac att gac att gat gag 2067 Cys Ser Pro Gly Phe Thr Gly Gln Arg CysAsn Ile Asp Ile Asp Glu 670 675 680 685 tgt gcc tcc aat ccc tgt cgc aagggt gca aca tgt atc aac ggt gtg 2115 Cys Ala Ser Asn Pro Cys Arg Lys GlyAla Thr Cys Ile Asn Gly Val 690 695 700 aat ggt ttc cgc tgt ata tgc cccgag gga ccc cat cac ccc agc tgc 2163 Asn Gly Phe Arg Cys Ile Cys Pro GluGly Pro His His Pro Ser Cys 705 710 715 tac tca cag gtg aac gaa tgc ctgagc aat ccc tgc atc cat gga aac 2211 Tyr Ser Gln Val Asn Glu Cys Leu SerAsn Pro Cys Ile His Gly Asn 720 725 730 tgt act gga ggt ctc agt gga tataag tgt ctc tgt gat gca ggc tgg 2259 Cys Thr Gly Gly Leu Ser Gly Tyr LysCys Leu Cys Asp Ala Gly Trp 735 740 745 gtt ggc atc aac tgt gaa gtg gacaaa aat gaa tgc ctt tcg aat cca 2307 Val Gly Ile Asn Cys Glu Val Asp LysAsn Glu Cys Leu Ser Asn Pro 750 755 760 765 tgc cag aat gga gga act tgtgac aat ctg gtg aat gga tac agg tgt 2355 Cys Gln Asn Gly Gly Thr Cys AspAsn Leu Val Asn Gly Tyr Arg Cys 770 775 780 act tgc aag aag ggc ttt aaaggc tat aac tgc cag gtg aat att gat 2403 Thr Cys Lys Lys Gly Phe Lys GlyTyr Asn Cys Gln Val Asn Ile Asp 785 790 795 gaa tgt gcc tca aat cca tgcctg aac caa gga acc tgc ttt gat gac 2451 Glu Cys Ala Ser Asn Pro Cys LeuAsn Gln Gly Thr Cys Phe Asp Asp 800 805 810 ata agt ggc tac act tgc cactgt gtg ctg cca tac aca ggc aag aat 2499 Ile Ser Gly Tyr Thr Cys His CysVal Leu Pro Tyr Thr Gly Lys Asn 815 820 825 tgt cag aca gta ttg gct ccctgt tcc cca aac cct tgt gag aat gct 2547 Cys Gln Thr Val Leu Ala Pro CysSer Pro Asn Pro Cys Glu Asn Ala 830 835 840 845 gct gtt tgc aaa gag tcacca aat ttt gag agt tat act tgc ttg tgt 2595 Ala Val Cys Lys Glu Ser ProAsn Phe Glu Ser Tyr Thr Cys Leu Cys 850 855 860 gct cct ggc tgg caa ggtcag cgg tgt acc att gac att gac gag tgt 2643 Ala Pro Gly Trp Gln Gly GlnArg Cys Thr Ile Asp Ile Asp Glu Cys 865 870 875 atc tcc aag ccc tgc atgaac cat ggt ctc tgc cat aac acc cag ggc 2691 Ile Ser Lys Pro Cys Met AsnHis Gly Leu Cys His Asn Thr Gln Gly 880 885 890 agc tac atg tgt gaa tgtcca cca ggc ttc agt ggt atg gac tgt gag 2739 Ser Tyr Met Cys Glu Cys ProPro Gly Phe Ser Gly Met Asp Cys Glu 895 900 905 gag gac att gat gac tgcctt gcc aat cct tgc cag aat gga ggt tcc 2787 Glu Asp Ile Asp Asp Cys LeuAla Asn Pro Cys Gln Asn Gly Gly Ser 910 915 920 925 tgt atg gat gga gtgaat act ttc tcc tgc ctc tgc ctt ccg ggt ttc 2835 Cys Met Asp Gly Val AsnThr Phe Ser Cys Leu Cys Leu Pro Gly Phe 930 935 940 act ggg gat aag tgccag aca gac atg aat gag tgt ctg agt gaa ccc 2883 Thr Gly Asp Lys Cys GlnThr Asp Met Asn Glu Cys Leu Ser Glu Pro 945 950 955 tgt aag aat gga gggacc tgc tct gac tac gtc aac agt tac act tgc 2931 Cys Lys Asn Gly Gly ThrCys Ser Asp Tyr Val Asn Ser Tyr Thr Cys 960 965 970 aag tgc cag gca ggattt gat gga gtc cat tgt gag aac aac atc aat 2979 Lys Cys Gln Ala Gly PheAsp Gly Val His Cys Glu Asn Asn Ile Asn 975 980 985 gag tgc act gag agctcc tgt ttc aat ggt ggc aca tgt gtt gat ggg 3027 Glu Cys Thr Glu Ser SerCys Phe Asn Gly Gly Thr Cys Val Asp Gly 990 995 1000 1005 att aac tccttc tct tgc ttg tgc cct gtg ggt ttc act gga tcc ttc 3075 Ile Asn Ser PheSer Cys Leu Cys Pro Val Gly Phe Thr Gly Ser Phe 1010 1015 1020 tgc ctccat gag atc aat gaa tgc agc tct cat cca tgc ctg aat gag 3123 Cys Leu HisGlu Ile Asn Glu Cys Ser Ser His Pro Cys Leu Asn Glu 1025 1030 1035 ggaacg tgt gtt gat ggc ctg ggt acc tac cgc tgc agc tgc ccc ctg 3171 Gly ThrCys Val Asp Gly Leu Gly Thr Tyr Arg Cys Ser Cys Pro Leu 1040 1045 1050ggc tac act ggg aaa aac tgt cag acc ctg gtg aat ctc tgc agt cgg 3219 GlyTyr Thr Gly Lys Asn Cys Gln Thr Leu Val Asn Leu Cys Ser Arg 1055 10601065 tct cca tgt aaa aac aaa ggt act tgc gtt cag aaa aaa gca gag tcc3267 Ser Pro Cys Lys Asn Lys Gly Thr Cys Val Gln Lys Lys Ala Glu Ser1070 1075 1080 1085 cag tgc cta tgt cca tct gga tgg gct ggt gcc tat tgtgac gtg ccc 3315 Gln Cys Leu Cys Pro Ser Gly Trp Ala Gly Ala Tyr Cys AspVal Pro 1090 1095 1100 aat gtc tct tgt gac ata gca gcc tcc agg aga ggtgtg ctt gtt gaa 3363 Asn Val Ser Cys Asp Ile Ala Ala Ser Arg Arg Gly ValLeu Val Glu 1105 1110 1115 cac ttg tgc cag cac tca ggt gtc tgc atc aatgct ggc aac acg cat 3411 His Leu Cys Gln His Ser Gly Val Cys Ile Asn AlaGly Asn Thr His 1120 1125 1130 tac tgt cag tgc ccc ctg ggc tat act gggagc tac tgt gag gag caa 3459 Tyr Cys Gln Cys Pro Leu Gly Tyr Thr Gly SerTyr Cys Glu Glu Gln 1135 1140 1145 ctc gat gag tgt gcg tcc aac ccc tgccag cac ggg gca aca tgc agt 3507 Leu Asp Glu Cys Ala Ser Asn Pro Cys GlnHis Gly Ala Thr Cys Ser 1150 1155 1160 1165 gac ttc att ggt gga tac agatgc gag tgt gtc cca ggc tat cag ggt 3555 Asp Phe Ile Gly Gly Tyr Arg CysGlu Cys Val Pro Gly Tyr Gln Gly 1170 1175 1180 gtc aac tgt gag tat gaagtg gat gag tgc cag aat cag ccc tgc cag 3603 Val Asn Cys Glu Tyr Glu ValAsp Glu Cys Gln Asn Gln Pro Cys Gln 1185 1190 1195 aat gga ggc acc tgtatt gac ctt gtg aac cat ttc aag tgc tct tgc 3651 Asn Gly Gly Thr Cys IleAsp Leu Val Asn His Phe Lys Cys Ser Cys 1200 1205 1210 cca cca ggc actcgg ggc cta ctc tgt gaa gag aac att gat gac tgt 3699 Pro Pro Gly Thr ArgGly Leu Leu Cys Glu Glu Asn Ile Asp Asp Cys 1215 1220 1225 gcc cgg ggtccc cat tgc ctt aat ggt ggt cag tgc atg gat agg att 3747 Ala Arg Gly ProHis Cys Leu Asn Gly Gly Gln Cys Met Asp Arg Ile 1230 1235 1240 1245 ggaggc tac agt tgt cgc tgc ttg cct ggc ttt gct ggg gag cgt tgt 3795 Gly GlyTyr Ser Cys Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys 1250 1255 1260gag gga gac atc aac gag tgc ctc tcc aac ccc tgc agc tct gag ggc 3843 GluGly Asp Ile Asn Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu Gly 1265 12701275 agc ctg gac tgt ata cag ctc acc aat gac tac ctg tgt gtt tgc cgt3891 Ser Leu Asp Cys Ile Gln Leu Thr Asn Asp Tyr Leu Cys Val Cys Arg1280 1285 1290 agt gcc ttt act ggc cgg cac tgt gaa acc ttc gtc gat gtgtgt ccc 3939 Ser Ala Phe Thr Gly Arg His Cys Glu Thr Phe Val Asp Val CysPro 1295 1300 1305 cag atg ccc tgc ctg aat gga ggg act tgt gct gtg gccagt aac atg 3987 Gln Met Pro Cys Leu Asn Gly Gly Thr Cys Ala Val Ala SerAsn Met 1310 1315 1320 1325 cct gat ggt ttc att tgc cgt tgt ccc ccg ggattt tcc ggg gca agg 4035 Pro Asp Gly Phe Ile Cys Arg Cys Pro Pro Gly PheSer Gly Ala Arg 1330 1335 1340 tgc cag agc agc tgt gga caa gtg aaa tgtagg aag ggg gag cag tgt 4083 Cys Gln Ser Ser Cys Gly Gln Val Lys Cys ArgLys Gly Glu Gln Cys 1345 1350 1355 gtg cac acc gcc tct gga ccc cgc tgcttc tgc ccc agt ccc cgg gac 4131 Val His Thr Ala Ser Gly Pro Arg Cys PheCys Pro Ser Pro Arg Asp 1360 1365 1370 tgc gag tca ggc tgt gcc agt agcccc tgc cag cac ggg ggc agc tgc 4179 Cys Glu Ser Gly Cys Ala Ser Ser ProCys Gln His Gly Gly Ser Cys 1375 1380 1385 cac cct cag cgc cag cct ccttat tac tcc tgc cag tgt gcc cca cca 4227 His Pro Gln Arg Gln Pro Pro TyrTyr Ser Cys Gln Cys Ala Pro Pro 1390 1395 1400 1405 ttc tcg ggt agc cgctgt gaa ctc tac acg gca ccc ccc agc acc cct 4275 Phe Ser Gly Ser Arg CysGlu Leu Tyr Thr Ala Pro Pro Ser Thr Pro 1410 1415 1420 cct gcc acc tgtctg agc cag tat tgt gcc gac aaa gct cgg gat ggc 4323 Pro Ala Thr Cys LeuSer Gln Tyr Cys Ala Asp Lys Ala Arg Asp Gly 1425 1430 1435 gtc tgt gatgag gcc tgc aac agc cat gcc tgc cag tgg gat ggg ggt 4371 Val Cys Asp GluAla Cys Asn Ser His Ala Cys Gln Trp Asp Gly Gly 1440 1445 1450 gac tgttct ctc acc atg gag aac ccc tgg gcc aac tgc tcc tcc cca 4419 Asp Cys SerLeu Thr Met Glu Asn Pro Trp Ala Asn Cys Ser Ser Pro 1455 1460 1465 cttccc tgc tgg gat tat atc aac aac cag tgt gat gag ctg tgc aac 4467 Leu ProCys Trp Asp Tyr Ile Asn Asn Gln Cys Asp Glu Leu Cys Asn 1470 1475 14801485 acg gtc gag tgc ctg ttt gac aac ttt gaa tgc cag ggg aac agc aag4515 Thr Val Glu Cys Leu Phe Asp Asn Phe Glu Cys Gln Gly Asn Ser Lys1490 1495 1500 aca tgc aag tat gac aaa tac tgt gca gac cac ttc aaa gacaac cac 4563 Thr Cys Lys Tyr Asp Lys Tyr Cys Ala Asp His Phe Lys Asp AsnHis 1505 1510 1515 tgt gac cag ggg tgc aac agt gag gag tgt ggt tgg gatggg ctg gac 4611 Cys Asp Gln Gly Cys Asn Ser Glu Glu Cys Gly Trp Asp GlyLeu Asp 1520 1525 1530 tgt gct gct gac caa cct gag aac ctg gca gaa ggtacc ctg gtt att 4659 Cys Ala Ala Asp Gln Pro Glu Asn Leu Ala Glu Gly ThrLeu Val Ile 1535 1540 1545 gtg gta ttg atg cca cct gaa caa ctg ctc caggat gct cgc agc ttc 4707 Val Val Leu Met Pro Pro Glu Gln Leu Leu Gln AspAla Arg Ser Phe 1550 1555 1560 1565 ttg cgg gca ctg ggt acc ctg ctc cacacc aac ctg cgc att aag cgg 4755 Leu Arg Ala Leu Gly Thr Leu Leu His ThrAsn Leu Arg Ile Lys Arg 1570 1575 1580 gac tcc cag ggg gaa ctc atg gtgtac ccc tat tat ggt gag aag tca 4803 Asp Ser Gln Gly Glu Leu Met Val TyrPro Tyr Tyr Gly Glu Lys Ser 1585 1590 1595 gct gct atg aag aaa cag aggatg aca cgc aga tcc ctt cct ggt gaa 4851 Ala Ala Met Lys Lys Gln Arg MetThr Arg Arg Ser Leu Pro Gly Glu 1600 1605 1610 caa gaa cag gag gtg gctggc tct aaa gtc ttt ctg gaa att gac aac 4899 Gln Glu Gln Glu Val Ala GlySer Lys Val Phe Leu Glu Ile Asp Asn 1615 1620 1625 cgc cag tgt gtt caagac tca gac cac tgc ttc aag aac acg gat gca 4947 Arg Gln Cys Val Gln AspSer Asp His Cys Phe Lys Asn Thr Asp Ala 1630 1635 1640 1645 gca gca gctctc ctg gcc tct cac gcc ata cag ggg acc ctg tca tac 4995 Ala Ala Ala LeuLeu Ala Ser His Ala Ile Gln Gly Thr Leu Ser Tyr 1650 1655 1660 cct cttgtg tct gtc gtc agt gaa tcc ctg act cca gaa cgc act cag 5043 Pro Leu ValSer Val Val Ser Glu Ser Leu Thr Pro Glu Arg Thr Gln 1665 1670 1675 ctcctc tat ctc ctt gct gtt gct gtt gtc atc att ctg ttt att att 5091 Leu LeuTyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Ile Ile 1680 1685 1690ctg ctg ggg gta atc atg gca aaa cga aag cgt aag cat ggc tct ctc 5139 LeuLeu Gly Val Ile Met Ala Lys Arg Lys Arg Lys His Gly Ser Leu 1695 17001705 tgg ctg cct gaa ggt ttc act ctt cgc cga gat gca agc aat cac aag5187 Trp Leu Pro Glu Gly Phe Thr Leu Arg Arg Asp Ala Ser Asn His Lys1710 1715 1720 1725 cgt cgt gag cca gtg gga cag gat gct gtg ggg ctg aaaaat ctc tca 5235 Arg Arg Glu Pro Val Gly Gln Asp Ala Val Gly Leu Lys AsnLeu Ser 1730 1735 1740 gtg caa gtc tca gaa gct aac cta att ggt act ggaaca agt gaa cac 5283 Val Gln Val Ser Glu Ala Asn Leu Ile Gly Thr Gly ThrSer Glu His 1745 1750 1755 tgg gtc gat gat gaa ggg ccc cag cca aag aaagta aag gct gaa gat 5331 Trp Val Asp Asp Glu Gly Pro Gln Pro Lys Lys ValLys Ala Glu Asp 1760 1765 1770 gag gcc tta ctc tca gaa gaa gat gac cccatt gat cga cgg cca tgg 5379 Glu Ala Leu Leu Ser Glu Glu Asp Asp Pro IleAsp Arg Arg Pro Trp 1775 1780 1785 aca cag cag cac ctt gaa gct gca gacatc cgt agg aca cca tcg ctg 5427 Thr Gln Gln His Leu Glu Ala Ala Asp IleArg Arg Thr Pro Ser Leu 1790 1795 1800 1805 gct ctc acc cct cct cag gcagag cag gag gtg gat gtg tta gat gtg 5475 Ala Leu Thr Pro Pro Gln Ala GluGln Glu Val Asp Val Leu Asp Val 1810 1815 1820 aat gtc cgt ggc cca gatggc tgc acc cca ttg atg ttg gct tct ctc 5523 Asn Val Arg Gly Pro Asp GlyCys Thr Pro Leu Met Leu Ala Ser Leu 1825 1830 1835 cga gga ggc agc tcagat ttg agt gat gaa gat gaa gat gca gag gac 5571 Arg Gly Gly Ser Ser AspLeu Ser Asp Glu Asp Glu Asp Ala Glu Asp 1840 1845 1850 tct tct gct aacatc atc aca gac ttg gtc tac cag ggt gcc agc ctc 5619 Ser Ser Ala Asn IleIle Thr Asp Leu Val Tyr Gln Gly Ala Ser Leu 1855 1860 1865 cag gcc cagaca gac cgg act ggt gag atg gcc ctg cac ctt gca gcc 5667 Gln Ala Gln ThrAsp Arg Thr Gly Glu Met Ala Leu His Leu Ala Ala 1870 1875 1880 1885 cgctac tca cgg gct gat gct gcc aag cgt ctc ctg gat gca ggt gca 5715 Arg TyrSer Arg Ala Asp Ala Ala Lys Arg Leu Leu Asp Ala Gly Ala 1890 1895 1900gat gcc aat gcc cag gac aac atg ggc cgc tgt cca ctc cat gct gca 5763 AspAla Asn Ala Gln Asp Asn Met Gly Arg Cys Pro Leu His Ala Ala 1905 19101915 gtg gca gct gat gcc caa ggt gtc ttc cag att ctg att cgc aac cga5811 Val Ala Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn Arg1920 1925 1930 gta act gat cta gat gcc agg atg aat gat ggt act aca cccctg atc 5859 Val Thr Asp Leu Asp Ala Arg Met Asn Asp Gly Thr Thr Pro LeuIle 1935 1940 1945 ctg gct gcc cgc ctg gct gtg gag gga atg gtg gca gaactg atc aac 5907 Leu Ala Ala Arg Leu Ala Val Glu Gly Met Val Ala Glu LeuIle Asn 1950 1955 1960 1965 tgc caa gcg gat gtg aat gca gtg gat gac catgga aaa tct gct ctt 5955 Cys Gln Ala Asp Val Asn Ala Val Asp Asp His GlyLys Ser Ala Leu 1970 1975 1980 cac tgg gca gct gct gtc aat aat gtg gaggca act ctt ttg ttg ttg 6003 His Trp Ala Ala Ala Val Asn Asn Val Glu AlaThr Leu Leu Leu Leu 1985 1990 1995 aaa aat ggg gcc aac cga gac atg caggac aac aag gaa gag aca cct 6051 Lys Asn Gly Ala Asn Arg Asp Met Gln AspAsn Lys Glu Glu Thr Pro 2000 2005 2010 ctg ttt ctt gct gcc cgg gag gggagc tat gaa gca gcc aag atc ctg 6099 Leu Phe Leu Ala Ala Arg Glu Gly SerTyr Glu Ala Ala Lys Ile Leu 2015 2020 2025 tta gac cat ttt gcc aat cgagac atc aca gac cat atg gat cgt ctt 6147 Leu Asp His Phe Ala Asn Arg AspIle Thr Asp His Met Asp Arg Leu 2030 2035 2040 2045 ccc cgg gat gtg gctcgg gat cac atg cac cat gac att gtg cgc ctt 6195 Pro Arg Asp Val Ala ArgAsp His Met His His Asp Ile Val Arg Leu 2050 2055 2060 ctg gat gaa tacaat gtg acc cca agc cct cca ggc acc gtg ttg act 6243 Leu Asp Glu Tyr AsnVal Thr Pro Ser Pro Pro Gly Thr Val Leu Thr 2065 2070 2075 tct gct ctctca cct gtc atc tgt ggg ccc aac aga tct ttc ctc agc 6291 Ser Ala Leu SerPro Val Ile Cys Gly Pro Asn Arg Ser Phe Leu Ser 2080 2085 2090 ctg aagcac acc cca atg ggc aag aag tct aga cgg ccc agt gcc aag 6339 Leu Lys HisThr Pro Met Gly Lys Lys Ser Arg Arg Pro Ser Ala Lys 2095 2100 2105 agtacc atg cct act agc ctc cct aac ctt gcc aag gag gca aag gat 6387 Ser ThrMet Pro Thr Ser Leu Pro Asn Leu Ala Lys Glu Ala Lys Asp 2110 2115 21202125 gcc aag ggt agt agg agg aag aag tct ctg agt gag aag gtc caa ctg6435 Ala Lys Gly Ser Arg Arg Lys Lys Ser Leu Ser Glu Lys Val Gln Leu2130 2135 2140 tct gag agt tca gta act tta tcc cct gtt gat tcc cta gaatct cct 6483 Ser Glu Ser Ser Val Thr Leu Ser Pro Val Asp Ser Leu Glu SerPro 2145 2150 2155 cac acg tat gtt tcc gac acc aca tcc tct cca atg attaca tcc cct 6531 His Thr Tyr Val Ser Asp Thr Thr Ser Ser Pro Met Ile ThrSer Pro 2160 2165 2170 ggg atc tta cag gcc tca ccc aac cct atg ttg gccact gcc gcc cct 6579 Gly Ile Leu Gln Ala Ser Pro Asn Pro Met Leu Ala ThrAla Ala Pro 2175 2180 2185 cct gcc cca gtc cat gcc cag cat gca cta tctttt tct aac ctt cat 6627 Pro Ala Pro Val His Ala Gln His Ala Leu Ser PheSer Asn Leu His 2190 2195 2200 2205 gaa atg cag cct ttg gca cat ggg gccagc act gtg ctt ccc tca gtg 6675 Glu Met Gln Pro Leu Ala His Gly Ala SerThr Val Leu Pro Ser Val 2210 2215 2220 agc cag ttg cta tcc cac cac cacatt gtg tct cca ggc agt ggc agt 6723 Ser Gln Leu Leu Ser His His His IleVal Ser Pro Gly Ser Gly Ser 2225 2230 2235 gct gga agc ttg agt agg ctccat cca gtc cca gtc cca gca gat tgg 6771 Ala Gly Ser Leu Ser Arg Leu HisPro Val Pro Val Pro Ala Asp Trp 2240 2245 2250 atg aac cgc atg gag gtgaat gag acc cag tac aat gag atg ttt ggt 6819 Met Asn Arg Met Glu Val AsnGlu Thr Gln Tyr Asn Glu Met Phe Gly 2255 2260 2265 atg gtc ctg gct ccagct gag ggc acc cat cct ggc ata gct ccc cag 6867 Met Val Leu Ala Pro AlaGlu Gly Thr His Pro Gly Ile Ala Pro Gln 2270 2275 2280 2285 agc agg ccacct gaa ggg aag cac ata acc acc cct cgg gag ccc ttg 6915 Ser Arg Pro ProGlu Gly Lys His Ile Thr Thr Pro Arg Glu Pro Leu 2290 2295 2300 ccc cccatt gtg act ttc cag ctc atc cct aaa ggc agt att gcc caa 6963 Pro Pro IleVal Thr Phe Gln Leu Ile Pro Lys Gly Ser Ile Ala Gln 2305 2310 2315 ccagcg ggg gct ccc cag cct cag tcc acc tgc cct cca gct gtt gcg 7011 Pro AlaGly Ala Pro Gln Pro Gln Ser Thr Cys Pro Pro Ala Val Ala 2320 2325 2330ggc ccc ctg ccc acc atg tac cag att cca gaa atg gcc cgt ttg ccc 7059 GlyPro Leu Pro Thr Met Tyr Gln Ile Pro Glu Met Ala Arg Leu Pro 2335 23402345 agt gtg gct ttc ccc act gcc atg atg ccc cag cag gac ggg cag gta7107 Ser Val Ala Phe Pro Thr Ala Met Met Pro Gln Gln Asp Gly Gln Val2350 2355 2360 2365 gct cag acc att ctc cca gcc tat cat cct ttc cca gcctct gtg ggc 7155 Ala Gln Thr Ile Leu Pro Ala Tyr His Pro Phe Pro Ala SerVal Gly 2370 2375 2380 aag tac ccc aca ccc cct tca cag cac agt tat gcttcc tca aat gct 7203 Lys Tyr Pro Thr Pro Pro Ser Gln His Ser Tyr Ala SerSer Asn Ala 2385 2390 2395 gct gag cga aca ccc agt cac agt ggt cac ctccag ggt gag cat ccc 7251 Ala Glu Arg Thr Pro Ser His Ser Gly His Leu GlnGly Glu His Pro 2400 2405 2410 tac ctg aca cca tcc cca gag tct cct gaccag tgg tca agt tca tca 7299 Tyr Leu Thr Pro Ser Pro Glu Ser Pro Asp GlnTrp Ser Ser Ser Ser 2415 2420 2425 ccc cac tct gct tct gac tgg tca gatgtg acc acc agc cct acc cct 7347 Pro His Ser Ala Ser Asp Trp Ser Asp ValThr Thr Ser Pro Thr Pro 2430 2435 2440 2445 ggg ggt gct gga gga ggt cagcgg gga cct ggg aca cac atg tct gag 7395 Gly Gly Ala Gly Gly Gly Gln ArgGly Pro Gly Thr His Met Ser Glu 2450 2455 2460 cca cca cac aac aac atgcag gtt tat gcg tga gagagtccac ctccagtgta 7448 Pro Pro His Asn Asn MetGln Val Tyr Ala * 2465 2470 gagacataac tgacttttgt aaatgctgct gaggaacaaatgaaggtcat ccgggagaga 7508 aatgaagaaa tctctggagc cagcttctag aggtaggaaagagaagatgt tcttattcag 7568 ataatgcaag agaagcaatt cgtcagtttc actgggtatctgcaaggctt attgattatt 7628 ctaatctaat aagacaagtt tgtggaaatg caagatgaatacaagccttg ggtccatgtt 7688 tactctcttc tatttggaga ataagatgga tgcttattgaagcccagaca ttcttgcagc 7748 ttggactgca ttttaagccc tgcaggcttc tgccatatccatgagaagat tctacactag 7808 cgtcctgttg ggaattatgc cctggaattc tgcctgaattgacctacgca tctcctcctc 7868 cttggacatt cttttgtctt catttggtgc ttttggttttgcacctctcc gtgattgtag 7928 ccctaccagc atgttatagg gcaagacctt tgtgcttttgatcattctgg cccatgaaag 7988 caactttggt ctcctttccc ctcctgtctt cccggtatcccttggagtct cacaaggttt 8048 actttggtat ggttctcagc acaaaccttt caagtatgttgtttctttgg aaaatggaca 8108 tactgtattg tgttctcctg catatatcat tcctggagagagaaggggag aagaatactt 8168 ttcttcaaca aattttgggg gcaggagatc ccttcaagaggctgcacctt aatttttctt 8228 gtctgtgtgc aggtcttcat ataaacttta ccaggaagaagggtgtgagt ttgttgtttt 8288 tctgtgtatg ggcctggtca gtgtaaagtt ttatccttgatagtctagtt actatgaccc 8348 tccccacttt tttaaaacca gaaaaaggtt tggaatgttggaatgaccaa gagacaagtt 8408 aactcgtgca agagccagtt acccacccac aggtccccctacttcctgcc aagcattcca 8468 ttgactgcct gtatggaaca catttgtccc agatctgagcattctaggcc tgtttcactc 8528 actcacccag catatgaaac tagtcttaac tgttgagcctttcctttcat atccacagaa 8588 gacactgtct caaatgttgt acccttgcca tttaggactgaactttcctt agcccaaggg 8648 acccagtgac agttgtcttc cgtttgtcag atgatcagtctctactgatt atcttgctgc 8708 ttaaaggcct gctcaccaat ctttctttca caccgtgtggtccgtgttac tggtataccc 8768 agtatgttct cactgaagac atggacttta tatgttcaagtgcaggaatt ggaaagttgg 8828 acttgttttc tatgatccaa aacagcccta taagaaggttggaaaaggag gaactatata 8888 gcagcctttg ctattttctg ctaccatttc ttttcctctgaagcggccat gacattccct 8948 ttggcaacta acgtagaaac tcaacagaac attttcctttcctagagtca ccttttagat 9008 gataatggac aactatagac ttgctcattg ttcagactgattgcccctca cctgaatcca 9068 ctctctgtat tcatgctctt ggcaatttct ttgactttcttttaagggca gaagcatttt 9128 agttaattgt agataaagaa tagttttctt cctcttctccttgggccagt taataattgg 9188 tccatggcta cactgcaact tccgtccagt gctgtgatgcccatgacacc tgcaaaataa 9248 gttctgcctg ggcattttgt agatattaac aggtgaattcccgactcttt tggtttgaat 9308 gacagttctc attccttcta tggctgcaag tatgcatcagtgcttcccac ttacctgatt 9368 tgtctgtcgg tggccccata tggaaaccct gcgtgtctgttggcataata gtttacaaat 9428 ggttttttca gtcctatcca aatttattga accaacaaaaataattactt ctgccctgag 9488 ataagcagat taagtttgtt cattctctgc tttattctctccatgtggca acattctgtc 9548 agcctctttc atagtgtgca aacattttat cattctaaatggtgactctc tgcccttgga 9608 cccatttatt attcacagat ggggagaacc tatctgcatggacctctgtg gaccacagcg 9668 tacctgcccc tttctgccct cctgctccag ccccacttctgaaagtatca gctactgatc 9728 cagccactgg atattttata tcctcccttt tccttaagcacaatgtcaga ccaaattgct 9788 tgtttctttt tcttggacta ctttaatttg gatcctttgggtttggagaa agggaatgtg 9848 aaagctgtca ttacagacaa caggtttcag tgatgaggaggacaacactg cctttcaaac 9908 tttttactga tctcttagat tttaagaact cttgaattgtgtggtatcta ataaaaggga 9968 aggtaagatg gataatcact ttctcatttg ggttctgaattggagactca gtttttatga 10028 gacacatctt ttatgccatg tatagatcct cccctgctatttttggttta tttttattgt 10088 tataaatgct ttctttcttt gactcctctt ctgcctgcctttggggatag gtttttttgt 10148 ttgtttattt gcttcctctg ttttgtttta agcatcattttcttatgtga ggtggggaag 10208 ggaaaggtat gagggaaaga gagtctgaga attaaaatattttagtataa gcaattggct 10268 gtgatgctca aatccattgc atcctcttat tgaatttgccaatttgtaat ttttgcataa 10328 taaagaacca aaggtgtaat gttttgttga gaggtggtttagggattttg gccctaacca 10388 atacattgaa tgtatgatga ctatttggga ggacacatttatgtacccag aggcccccac 10448 taataagtgg tactatggtt acttccttgt gtacatttctcttaaaagtg atattatatc 10508 tgtttgtatg agaaacccag taaccaataa aatgaccgcatattcctgac taaacgtagt 10568 aaggaaaatg cacactttgt ttttactttt ccgtttcattctaaaggtag ttaagatgaa 10628 atttatatga aagcattttt atcacaaaat aaaaaaggtttgccaagctc agtggtgttg 10688 tattttttat tttccaatac tgcatccatg gcctggcagtgttacctcat gatgtcataa 10748 tttgctgaga gagcaaattt tcttttcttt ctgaatcccacaaagcctag caccaaactt 10808 ctttttttct tcctttaatt agatcataaa taaatgatcctggggaaaaa gcatctgtca 10868 aataggaaac atcacaaaac tgagcactct tctgtgcactagccatagct ggtgacaaac 10928 agatggttgc tcagggacaa ggtgccttcc aatggaaatgcgaagtagtt gctatagcaa 10988 gaattgggaa ctgggatata agtcataata ttaattatgctgttatgtaa atgattggtt 11048 tgtaacattc cttaagtgaa atttgtgtag aacttaatatacaggattat aaaataatat 11108 tttgtgtata aatttgttat aagttcacat tcatacatttatttataaag tcagtgagat 11168 atttgaacat gaaaaaaaaa a 11189 5 25 DNAArtificial Sequence PCR Primer 5 tggcaactaa cgtagaaact caaca 25 6 23 DNAArtificial Sequence PCR Primer 6 tgccaagagc atgaatacag aga 23 7 37 DNAArtificial Sequence PCR Probe 7 acaactatag acttgctcat tgttcagact gattgcc37 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 920 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 121434DNA Homo sapiens 11 tatcctccct gttttctcac ctgagagtgg gtataatgaaatttacttct aagggttgtt 60 gtgaagattg aattagttag ctaatttatg taaagtacttaggatagtgc ctggtattta 120 gtaagttcta tgtgttagcc tattattatt attactattaccatttaaaa atattatttc 180 aaggatggga tgcattttct gtaaacatgt tctcaaatgattctactact ggatacaata 240 ctgggctttt atgcagtaat tgttgttgga ggagttgttctgacatccag cttgcacctg 300 ggctactggt ttcgtggcca gggatggcca ggcctagcgtctctctgatc ttgcacaatt 360 tatgcataga gtggtgtggt tagaggaaac ttggagagggggaacttctc agttttgctt 420 tcaggtattt atacattaac tatcataaga gatggacatcttaataactt ccaagaaaga 480 gttcatgagc ttcaatttga tatttattgt ttgactaggtttctttctgt cttacctttc 540 cagttgttag ggttcttgat atgtattttg gtgatgaaaatttcaactct aaaagctaaa 600 caatcaagat attgtaatta tccagcaagt gattggcagatttcgagtag acttcttcca 660 ttcttggctg aaatcaatga aaatgtctac tattgctgttgtttttgttc ttttctctga 720 aaagtctgtt tttgggaggg cctcagatgg taaagtgcctttttattgtc tcttaaaggg 780 aatgggggaa tcaacagttt gagaattttt gttttatctgtatgtataaa attgccttct 840 ccttttattt cttttgatac tctctctgca gtctctattcatgatctact ccccatcttt 900 ttatgggccc cttaaagcta gagattatgt ttattcgtttatttatgtat tccatttctc 960 cttttttctg gatcctaatt ttgtgcctgg tgcatactaggctctcaatt ttagtttgtc 1020 gaattactgt tattgctgtt tctgattact tcttgaagcggtctgtttta aaacttcacc 1080 tgaaggctaa gtaattcatc agtataatgg tgatctattatttatctagc attttcctaa 1140 acactcattg atgaatcctt ataatactca tctgaagtatctaaatccca agatgatgtg 1200 tcatgccata gatggtatgg ctttaatttt taattgggagatggagatgt aacagctgga 1260 gatactaaaa cacagagaaa taagagcatc attcaagaccacaaacgtgg ttattgttgg 1320 aagatagttt tgggatgggc ttctgtgcta gaagccagaacataaaaaac tgattaaaac 1380 tctaaaaaga aacccagatt aggtgggaat gaatgctgatctggattgga ttacacttct 1440 ttggtgtctg agggttatga ttagtgactt acttctaatgtgcatttgtt tttattttta 1500 gcattgcagt gtcgagatgg ctatgaaccc tgtgtaaatgaaggaatgtg tgttacctac 1560 cacaatggca caggatactg caagtaagtt tttctcttcatatattttct ttttgcgata 1620 gaacactgga caagatttga ttctactcct ctatttttaatgcttctgtg gaatgttact 1680 ggttcttgag ctttcctggt acagattttg gttgggggtgaggatggaag gatgtggatg 1740 ccagataatt ggctaataag aacttcttac tatcttactgttatctttct cactatgaaa 1800 aagacttttc agtgagtggc tagttaatgt atatggggtagaccaggcat ggtggctcat 1860 gctggtaatc ccagcacttc aggaggctgt gttgggcgaatcacgaggtc aggagttcaa 1920 gatcagcctg gccaacatag tgaaacccta tctctactaaaaatacaaaa attagccagg 1980 tgtggtggcg catgactgga gtcccagcta ctcgggaggctgaggcagga gagtgacttg 2040 aacccaggag gcggaggttg cagtgagctg acaccacatcattgcactct agcctgggtg 2100 acagagcaag actccatctc aaaaagaaat aaaaaaaaatataaaaaaat atatattaga 2160 ttcccttgtg tttttcagat taaggcatac tcttagagtctccccttaac atgttaccta 2220 ttatcttgtt tttaagtaaa accaaataga tatcaactttaatattcggc ccagtatatg 2280 tgaatattat attcatattt ttttctagtg taaaaaatctaattctgaaa gtgagacagt 2340 gagagaggaa gatgtgtgca tgtgtgtaga ttgagggggagcaggggctt tgggtgtgta 2400 ttattatatg gggataaaag agtaaggatg aaggaaaatgtaggctagtt cttggcttaa 2460 gagcattttt ttttcctttt tctccaacca tctactctagccaagacgtt ttgtttgttt 2520 gaggaaggct gtgagataat ttcataatag cttttctaggtttcctgcaa ataatttttt 2580 tttcttctgc ctcaattgaa aatttttaag aggaatatgtatccattctt tttatttcag 2640 ggaagatgct ggagaaataa aatttggtaa tatgaaatttctcttctttt ccttcatttt 2700 tgtgaggagt agttgttctt tgctttggtg gaggtacttgcttagtaagc attttaagtg 2760 agtttatcca acacattttt atttcttacc aggaatgtaattacaacttt tttccagtga 2820 gatctgttct gacaccagga tttagttttt taatgttataaacaagattt tttttttcaa 2880 gtcagaaata attttcttca ctaaagtgaa aatattttaagctgtgatga cagtaaagct 2940 taacaatagg ttgtttggat tggaataaag ataacattggaaataaaggt tttatgtagc 3000 ttattatggg ctgctcattt agtttttcta gctgggggaaaaaaaaacgt ggtgcattct 3060 cctctaagaa tggagataca actggagata ataagggagggaacttaata ccttagagta 3120 ggccactgaa atcttgttta gtctttttgt ggcatttggtgccttagttg cttgctttat 3180 tctgttatgc aactcttgtg gtagttaacc ccattgcattatggtcattt gttgatgtgt 3240 ttcttttgct agaatgtgag gtcttagtct tatgcattttttgcatagtt aatgcctagt 3300 acagtgcctg gtgtagttat tgttcagcaa tgtgtttcttaaactaaaag gtgctaagta 3360 gataccatca tgcacatgtt tcttatctat ttatgttaaataagaagaca ctggttctct 3420 cctttaaaaa tttcagtgtg gcgattcctc aaggatctagaaccagaaat accatttggc 3480 ccagcaatcc cattactgag tatataccca aaggattataaatcattcta ctataaggac 3540 acatgcacat gtgtgtttac tgcagcacta tttacagtaggaaagacttg gaaccaaccc 3600 aaatgcccaa cggtgataga ctggataaag aaaatgtggcatgtatacat gatggaacac 3660 tatacagcca taaaaaagaa tgagttcatg tcctttgcagggacatggat gaagctggaa 3720 gccatcattc tcagcaaact aacacaggaa cagaaaaccaaacacggcat gttctcactc 3780 ataagtggga gttgaacaat gagaacacat ggacaaagggaggggaacat cacacactgg 3840 ggcctgtcag ggggtggggg gaaaggggag ggagagcattaggatgaata cctaatgcac 3900 gtggggctta aaacctggat gacgggttga taggtgcagcaaaccaccat ggcacatgta 3960 tacctatgaa acaaacctgc acattctgca catgtatcccaaaacttaaa agtaaaattt 4020 aaaaaaaatt tatttacttc acatagcatt cagggtggtaataccataca gagaggcaaa 4080 ttggttctat ttgaagatca ttcaacttgg tgacttatatatggagaatt tcaaatatgt 4140 tctaagtgtt cgcctatata tttttggtct gacatggggaaagaaggttt ctaggggaga 4200 ctaatctctg agcacttacc aggagaggac acctaagggtcactgttggg aagaagtcca 4260 atagttcttt gttggactgt tgggctgcta gtctcatactctccaaccaa ggttggagac 4320 catcctggct aacacggtga aatcccatct ctactaaaaatgcaaaaaaa ttagctgggc 4380 gtggtggcac gcacctgtaa tcccagttac tcgggaggctgaggcaggag aatggcctga 4440 acccaggagg cggagcttgc agtgagccaa gattgcaccactgcactcca gcctgagcga 4500 cagagcgaga ctgtgtctcc aaaaaaaaaa aaaaaaaagaaaatggagct gcctaaattc 4560 tgtccctggc tacttgttaa ttgttgatag gatggaatgcttatggatac agttctgggt 4620 atgtccctca gatgtctttc ctgttttgag ggaaacaaactccttgagag agagtaagaa 4680 aaaatttggt gaggccatcc ttttgggaga ccaagatgaacaacaaaact tgtataaaga 4740 gagtgattcc ctacctcagc aaaagtaata ttagcatatttaggagtggc agggtcaaca 4800 taagtggaca aagaaaaagc aagaacaata tcaaggcagccatgtcaatc attctacagt 4860 gctttgccag cctccacaga attttaagca aaattttatgcaaaaataat gtgtacctag 4920 cagagctttc agtgtatttt caccaacctt acctttttttctgtggagaa agaagatgat 4980 ctagcttgtt cagtaaatat tattttcaca aaatagaaaataaatattaa aacacttaga 5040 agatttataa aaattctatc aggcatgcag ggcatgtgattatcttttta ttcatgcgat 5100 tgcttagtgg attaagaaaa gaaagttgaa gccaaattccttttacaggg ccagcgtagg 5160 ggtgagtggt gagtggtggg atgggaactg gtagaaagtgttttaaaata gaaggaatct 5220 gactatctgt ttagacagca taatgctact gattcagctaaaatataatg attgtttata 5280 gaaattataa aaatccttga taagatcaca ttgacagatgaagataacta cttttgaaga 5340 ttagttttgt actcagctcc actgtttttt ctcactgttaggagcctaag taccctatca 5400 gtctgtgatt agattttgat gccagattag gagaacgtgtataatcttgc ataaaactca 5460 attattgctg ttaaataact actgtcaaat cttaataaagctgtctcact acagggagat 5520 aagatatttt attctttccc ttgtcctact gggaggtgtaaatatgtaat taaatttgat 5580 gtcattaatc actttcaagt tgtttgcttc agactttcaaatataaacca ttctaaatcc 5640 caatttgatt aaagatcatg gacaactcaa gttcactaggattctggggg taggttaggt 5700 tatgatttgt ataattgaca aaatatgtat tttaaggacatttattgtag ttatttaaaa 5760 tctttgtctg ctggttctta acatctgggt tgtctatggatgtgtttctg tttctatcaa 5820 ctgctccttc tttggaccat gggtatgact tccctttactttacacatgt atggtagttt 5880 tacattactt actggacatt gtgggtgata gattttaaagtttatctccc actaaagaat 5940 gttgaatttt gtcaagattg gcagatcatc ttgatcctatggatgctagg ttttaggaca 6000 cgttagggtg ggcctacttc tgttctgccc ttagtccgatgacacaaatc ttagtcatgg 6060 gatagggttc ttattcccaa gatgtgaccc ttctaggggttcagtggtaa gtgtgagggg 6120 cctaccaagc cctgaggtgt tcattaagcc cctctaaggcagaacttgtc ctcccagccc 6180 caggagcctt ctgctcagtc ttttcagccg tccagctcttactttctctt gggtttctga 6240 atcttatctt gctcatgcat agtcaggaat tatttaaggatttgagcaga gttgtatgca 6300 tactttgaag ctacccactc tgtggttctg tcttttccaggaatttttct cctcatattc 6360 tagctgctgt gacagctcca gatacctctg actcctcagtatcacaggac ttccattttc 6420 tgcttgcaca ctcttcctct tctgctgcat gaactggggtgtaccttcac gataaaaaaa 6480 aaaacaaaac tgtaaatgtg ggtttcatat agtttgctttgctttttctg aggcttatat 6540 cccttctagt ttatgcctgc tttcagtgat tctccagtgcctttgatatt ttgtccagag 6600 caagagggct agtctgatat aagttactct gcaattatttttaatgatga aaattggaaa 6660 tataaagggg cctttcagaa tttcatacat accacttggtaaatatctaa cgtagtgttt 6720 gtaaaacagc agaaagtcac tgtgagttgt atacattcctgtagatgggt gcttgcattt 6780 gagaatgtcc actttttgct aaagtgctga tctaaaaactacactttaag gtaaattatt 6840 ataaatataa agtgttataa aatataactt tgtgtcacattttgtctaaa acttgtctgc 6900 tagctgtaat aaacattaaa cattaacagc taacaattttccttgccagg attaggcagt 6960 catgttggtg gtccagattt cctgaatcca tccaagaaaaactagagcca ttgccttctt 7020 tgtcttcttg gtaaatgtct gtatagtaag tagagagtagagacactcat aatcccttag 7080 aacttagctt tttatggagg atgcattcgc aatggttctgttgtggtgct tctcccccaa 7140 aagggcgttt tcagaagtca tggctgactc agcatttccccttttccttc accttcttgt 7200 gagtgcattc agggaggcac aggggcattg tcagatcaaagaaatagaca gggaaaaata 7260 tcaactgtta aattactttc tctttcttct cagctgaaactggttctgcc agcctagttc 7320 cttcaagtac ggtgcctatt tagctgaaca ttgtgtatatagtttttctg gaaaatgata 7380 gcagaagctt ggccagtttt ccatagttca tttatctttttaacataaaa caaagaatgc 7440 tgttttcttg gctcattaaa tacctctcat agagtaatcttttcttaaaa tgtaatgtgt 7500 tcaggtttta ttaattcaag agttatttat tgcatgctatttatgagtac cacatataac 7560 agataactca agtctgtggt agtagttgca gttaatcaatattgttatca tattatttca 7620 cacttctttg ggaagtcata aaataattac cagttcattaacttgggctt cttagagagg 7680 ttcagagtaa agccaagtat gaaaaaaact gactataatgtggccttctg tacaggggcc 7740 gccagtgaat ggcagggctg ggaccatggt gaggcaagtaaggcacaggc ctctcttgtg 7800 aaatttaaga gagtgccaaa aaactcacta atcaaaataatgtttgtatg tagttgttca 7860 aaaaatcaaa attaatgcaa aataagttga aaattatcaaaattttaaat aaggagataa 7920 gtactactga ttttttcttt tgccttatga tctagtatggttcagcctgc actggtgagt 7980 ggagtaaggg tcctggaaga tccttgactg gtgacatgaagaggacatag ttttttgttg 8040 ttgttttttt ttgagacaga gtctcgctct gtggcccaggctggagtgca gtggcgcttt 8100 cttggctcac tgtaagctcc gcctcccggg ttcttgccattctcctgcct cagcctctca 8160 agtagccggg actacaagcg cccgccacca cgcccggctaattttttgta tttttagtag 8220 agacggggtt tcactgtgtt agccaggatg gtctctatctcctgacctca tgatctgccc 8280 gcctcggcct cccaaagtgc tgggattaca ggcgtgagccaccacaccca gctgaggaca 8340 taggtttttt tatgccagat agacttaagt ttgagtcctgattcctatta acaattggat 8400 ctttggcaag ttaaagtcta taatgctagc taacattattgcgtattagg gtctgttctc 8460 aagtagttta acatgtattt actttagtcc ccatgtgagagagagactat atggaagcta 8520 agagaagtta aataattcac tcaagatccc atcactctgtagctaacctg agatatactc 8580 gtggaaaatg tactgtcacc ttgagtctca gtttgcctgtcaataaaatg ggacagaatt 8640 acctgtttta agtttgtagt tcaaatggaa ccttctgaaaaggaagtgtg gaacatttga 8700 aagtatacaa aaaggggact tttcagatca ggaatatgattaaaacactg ctttatgtaa 8760 tttctaaatt taaagacagt agcattaggt aggcctgagtacctcatatc attctagaac 8820 actaaatctg catatagtta catgagtgta catgggccaggtttggtgaa tactgcattt 8880 ctgagaaatg gcctttaaca tgaaagctca atgtaataactgtggatgaa atagaagaag 8940 agggcagtga aggtggggat agaggatatt gtctattaaagtgtgcttca gtagagagaa 9000 ctaatttgaa acttctcctt tctttttact aatggaacaggagcaagtcc agacagattt 9060 atgcagttgt gaaattgaga caggttaaat tcagtgatttttgataaatg ttgttgccag 9120 cacaaaagtc acaagttgtt gaatatggca ccttacatttgcatagtgag ttacagatgc 9180 atatgagtat gtcctgtgag gcttttcctt gtattaggacttgagatctg gaccctttaa 9240 cagtaccatt cctaagtgat tcttattaat acttatgtcctattaaaatc ctttatcaaa 9300 tatgatgttt ccctaagggg tcttttgtaa aggaagtcttggttactagc atctccatct 9360 gtggcccagg cttgagtgca gtagagcaat catggatcactgcagcctcc acctcctagg 9420 ctcaagccat ccatcctcct gcctcagtct cccatttttaaaacttttta tagagacacg 9480 gtctcacatg ttgctcaggc tggtctcgaa ttcctgggctcaagtgatcc tcccacttct 9540 gcctcccaaa gggctggaat tacaggcctg aaccactgtgcctggccagt tagtagcttc 9600 ttaacacata gcaacataat ttccatacta ttttcaactaatttttattc tttcagttgg 9660 gacctttata tatttcagtg ttcagtgaag aagtaggagaatctggttct taggctctgc 9720 ctatatcctt ttacctgtct tgggtaactg ccaagaaattaaatcaacaa attttggcct 9780 gcagtctggc attaatagat gtgaatttag ctaggtttctttcaacacag tctccaggtc 9840 aatcgtaatc ttatgtgaag tttcttattc aggcatgtctcattagcacc attaatttca 9900 cacaggaatt ctttttttct cccttttttt gtgactaagcaaaatgagaa atatttctct 9960 ttcaggatat ggagaggctt aattaaattc tttaattttgtctacatctg cccttatgaa 10020 tgatagcaaa ggaatggggg atatcagaat atgagaaaaagtaatcatct aaaaatttcc 10080 aagcacttta aaatttgtta agctagcata aatatgtgggtgtgatagag gttttcatta 10140 ggcctcctct caacatacag tagttaggaa tgaagtttagtgtctctccg tcctcctgtc 10200 atatgtgtgt atgcacacat gtgtgcacat gcgactgtgttatgcatttt gttttcatat 10260 ttcttttagt tgagttttac cttacaaccc aacgtaaggtaaattaaact ttctattgga 10320 tttccgcaca ttttctagat cctatttatt cactgtcagtgaccttcact agcacttagt 10380 cctctgtgtc caaaaatgta ttgccaattc ttagactccatcagccttct cctatgaaaa 10440 agtaatgcag ttaatttctg ctcgaaatgc tcttctcctaaacagtcttc tacttgatct 10500 ttatgaaaag agttttatga aagaaaagtg aaaataaagactaagaaagg cattatttaa 10560 gtgatgggtg atattattca gcgaacacat tcaactagagcattctatcc agttttttgc 10620 cttcacgtaa atatgaggag aaaggtaggt gttaataagaaaataatctg attaggattt 10680 atgtagaatg gtagcattta gactattaaa tctgtatagattcaggtcat tttcctgcag 10740 tcactgtcta taatcctgtg tgctgaacac tgaaagttagatggactctg aagcactata 10800 gaatcctcac ttttcaaaca aaagtttaat ttttatcacactagtattta ttgagcaggc 10860 tactataact gatcactgta ctgggtattg tgaatacccagaaaagccca agaggcctgt 10920 gttacctgac ttccttattc acaagtatac gtacccagagactgtctgac agtgaatgca 10980 gtttctcctg cataagacat ggtggcagtc ttctgaagtcttgctgggga gatgaagcat 11040 acagagatgt gtaaaataaa acagtttgta atatcccagtgtaggggcac agataataac 11100 tatggacact cagacaaagg aacaagtact gtgggacaaaagagtaaaat cagaggactt 11160 ttggtgcact gcttagatcc taaatctttg aatattagtggtgccttaga agacggatag 11220 gtaggagaag ggtgagagga agggagctga ggagactagaagggactgtt atttcctgga 11280 ataatcatta ccaatttctt catcacattt tcttaatgacacagtttcaa acgttttctt 11340 catcttgatt gctgtcttct gtgtatatgc tggtttgtgtgaggttttac taaaccagta 11400 tgtggattaa tctagtttca cagtgacagg taagtgatttagaaaatcct tcctttagca 11460 catgttaact tgaacgtttg gaggtagaat gcctagagtgagggtgaagg agaggacctg 11520 gggacaggaa gagagggcag ctgggagcaa ttatgctccttccctcagac ctttttggcc 11580 ttgactcttg attcttgtac ttctaccaga cagagaggcggtaggcttgt ggccatgttt 11640 tgcgaggaaa atcagaatct aagaatctct aaggcaagagcaagaatgga ttgggatagg 11700 gaaagtgata ggaaaacaga ggcactaaga agagggttggttttcatgta ctgcatagtt 11760 cattccctta gttgagaaat caggtatctc aacctcagatatcttctgtc cagcatatcg 11820 ctggccagat ctgattcata tgagggatcc agagtaaggaagcatctgtt agccaagtct 11880 ttggttaccc caaattagct caggtccttt actgcttgtttcctctgatt atatagtaca 11940 tatccaaacc ctcaattttc cagaataaac taggtctgcctattctgtgt ggaaaacatt 12000 tgatggtggt taagattctc ttgcctagac gatggtagtgccttagtcgt ttaactgcct 12060 gtcactttaa gattttgaaa tactggttct cgttgtgtaagccagacaca tcttgagaat 12120 attttacggg gcaacttgcc ctttctgaaa cctcagtcatcctgaaaagt aactctgtac 12180 ctccttgact ttaatttttc tgtcttaatt ctatttgtatacaatgtttg tggtgtctga 12240 ttctctgtct cgtattgctg aaactcatgg ccttgaatttctgggtcact cttaaaaaat 12300 agacataact tgattactaa tgaaaaagtg actcattttcttccaggcaa aggctgacac 12360 tgactgtcac agtttgtgac agaagcaaac tctctaattaggagacatca cctattcttt 12420 tctaaatttt ataagaattg atgatagcat catgagatgggaagtgagta agataagcgt 12480 ttatgcttgt agagaggatg cataaaaatg aaatctggataaagttctat ctgaatttac 12540 cattttaagt ggttgattcc ctgtgagctt aaatttagtgcttctcttcc cagaccgttg 12600 tcttacatac acacttcagt cagttttagc cttgtataactaatcagcag gtcagattcc 12660 atagaggctg aaatacctac caccattcta tgctcaagaaaacaggctct catgttggcc 12720 agttgtttgg gatagaagat ggttagacct ttctatcccttttaaacaca agattctttt 12780 tcattcccct tatcttattt aaatatattg ttccttctgtttggttgttc tttgctttct 12840 tatgcatctg tgaggggcta gctgaaagtg tgagttgtggaacaattcat ttacataagt 12900 gtatattgaa ttatctgtgc acctagaggg tgatgggcactgtgagggat attaaaaaag 12960 agacacagta cagttcttac cctaggatcc cctgcttggagtgggacagt ggtcaagaca 13020 tatacaacca caaacaccca aacattaaca tcagcaagcatcattacatc cttgggtggt 13080 attgccaagg gacttcaaag gccaagatta tagagtcatcacacttagga ggtcaaaggg 13140 actttggaaa gcacctttgt cactcatttt gttttataagtgaggcggct gaggtgtaat 13200 acagctaagc gaattagcct ttattacata actcattagaatgtagactg ggactaagag 13260 ctgtttctcc agatcccatt aggcaatcta aagattctgcactatttctt tttcattttt 13320 ggtatccaaa taggtacagt tgtttttgat ctaggaaaatccttttagac taaacagtaa 13380 gctttttaaa ttactctgct tttagatatt ttttggacttcagaactttg aaggtggtca 13440 tccaccaaca ttggggaatg gaaagaaggc tccaacacttttctttctag agatcttctt 13500 taattttcaa tggcagtcac tacagaatct agcagttcatgctattgagg ccactttgta 13560 ggattttttt ttttcttagt cagggctcca ggtccctggtggtttatgta gtcagtcttt 13620 tatcaacaac cgatgaaaga ctaataaaag tgcaagagggaatgaggact gtgtgtgtgt 13680 atgtctgcac atgcacacat gcctctctgt gtgtgttggttaacttcata tttggtaggt 13740 gggagggaag gtgattaaaa aataatctac tttgcatggagttggcccaa tttgactgat 13800 ataggggtga gtgggggtta agaagtagtc tatatatcaggaatcttaat tagttttgaa 13860 aatcctttag gtaaatagct caacccagtt gttcccaaacttcaagtgta tttatagggt 13920 ctttttaagg ggtaaagatt ccacagagcc cttcattttgaccttgtgct tctgcactga 13980 atagttgaat gccttctata tgccagtcat tgtgttaggcactgggagta tgagaattaa 14040 taattttagt ctgttgacag ataatcataa taagggctaatgagtaacac aggctcaggg 14100 ttataagaac agagggggta tcaaggctgt ttttcacaggaaataacatt taagttgaga 14160 cttaatgaaa ttttagggct tagccatgag aaagtggggaaaagcatttt gggctgactg 14220 aacagcgtgt acagaggtct ggaagtgaga agtagtgtactatagcattt tctaggagct 14280 gaaaggcgtt tagcaagagt atagtgcata aaggagagagcttgagatga aacaatttct 14340 cagtgtgttg tttgaataca atgtatactg tcaaaatctaagaattcaag aatcatttaa 14400 tatatcttta tgttttacta gtcataaaag catatacatatatttaaata tggacaagat 14460 actgtttagt agtttagaaa gtgatttata aggattgcaatcccttgcaa taactccaca 14520 gcccatacct tgggaacaac tgagctaact cattcagttttgtccattac atcgaagggt 14580 gttttttgta aactcatatg tctagaatgt gtttgccatgttttaatcac aagtagatat 14640 tttctcaggg tccagtgcag tgaagtatgg taagatcctgattgtgttct ggaacacaga 14700 ggaaagacca ccttctgtta tagcaacaac acaagtcttttaacactgtg tgccccttcc 14760 caatctttca agtgatgatt gaagagacta ggtgctcagctcagcctttg agttctgata 14820 aatgagccca gactgtaaac tggaagataa ggatgtttgtaaagttcttg tataaataaa 14880 gcatggtttc tcattgcagt ggttactgat ttcatagtctgagtgaagat gaatgatgct 14940 gtgaatcaac agctttaaag tccgtatcac ttcagcttctttttggttta ggtttcttaa 15000 aatcagtgtg tatttaatgc tttattcaga tgagggggtgaaaaacctaa cacatgtaaa 15060 ctaagtgagg tggggtttca gagataattc ccagcctcacaattcctcgt gaagttcttt 15120 tcctgtggga aacttttaat ttggaagcat gcaacctaatgtgggaacca agattaacat 15180 tttctgaaat acttctacaa gaaaagcaga aatggtctgtccaggaagct gaatttacat 15240 agtagaaaaa tgagctgccc tgcagtattt ggtagtctttgtgtattagt tgtgataaaa 15300 gtgtgtatgt gtgtgtgtac gtgtgtgaga gtgagagattgtatacttgt ctttgtttcc 15360 ttcacataca actagtaagg ccctagaaaa actacactagaaagtgtgtt ttaccacaag 15420 cgtcccagtt ctggacacca atctatacac aaatacttttttttaaagtt ctttttgttt 15480 ttccttcttg ctgagtaagc tatagtattt cctttttttctttctttttt ttttgagaga 15540 agggggggtt gagagtagag tgggaatggc aagaagtagtatgacagagc ttcttctctt 15600 tttttcccct ctttaccagg aagttaacta gaagtcctcatgcatgtttt taaaacaaag 15660 ttggtaatta gcataaccta gttagttacc tttacacagagtgacagaat taaaaagttg 15720 acaagcccat cagacctcag ccaggaggta ctgaaaggagggagaccagt gagtttagac 15780 caataggtgg gttaggcctc ctgaatgcca gcctagaagtttagacttga ttctataggc 15840 tctggggtac ctacaagttt gtagtcggag ccttgggaattgaatgttac ataggaactt 15900 tcactggttc cagctagcct tggctgttag caattatttttatctacttt aacagggggg 15960 acagagtagg ggggcaggaa actaagctgg cattatggtcacaggaaaga acagactgat 16020 ttggagcctt tcaaactgca gacctttgtt actgaccgatgcttaatttg gtttctgggt 16080 tttgttagtt ttttcccctg cccttacctc atttaccttaacgacagctc ccccctctag 16140 agctcagcta gggcaggctg ccactgcgga ttggggggccaagaggccca gcgcaagaag 16200 aaagtgggtt gaaagcagag ttctgttcaa agaattttctgctggaaact agcccagagg 16260 gagtaaagag gagctttaat gaggagcagc tgcagtgccgacgcaaccca catgagactt 16320 tttttcccct tcgttccaca ttctgtatag tttttttaaaaatcatgatt ttgaaatagc 16380 tgttttgtaa agcatgcctc tctttttctt cttgtatgtggtgggatttt gctttgttgt 16440 tgttgttgtt tttttttgaa tggccaaatc ctcgtttttaaaaaaaaaaa aaaaaaagct 16500 aaagacagag ctgcagcaaa gccctggatg caatttggcctcaccctgct gatacagaac 16560 attcggtgga gaaaacaagg ggagagaaca ctggcttttatttggaaaag gggcttattt 16620 cctgctcaga cttcagtcat cttggagctg acacaagctgctacactgtt ttaagctttt 16680 ctgtagacga gtggctattc acttaggaaa cgtgaaagaacaaatttttc tgtcctgtat 16740 tactagggag actgatcctg aactgcagcc attgccagatagattggaat tgctattcag 16800 atcccagctt cgttgaaatc tgtaaagtgg ctacatgtaaactaatccag gctgctagtg 16860 agatgtgagg gttggggtct ggttttcatc tgcttaagtgagaggaaact gtaggggtat 16920 ccttcaaatg gaatgttttc tagttccatt aggaggaatcccttgttttt ctctgttttc 16980 ttccttcctt tgcttttcta catccaaccc catgcgtatgtttgaacagt aacaatggaa 17040 atgtggacct ctcaatgtca gaaatgacac ttttttttttttgagatggg gttctcacca 17100 tgttgcccgt gttgtctagg caggtctcga acttctgctctagcaatctg cttgccttgg 17160 cctcccacag tgctgggatt acaggtgtga gccactgtgcccagctgaga cttttaaaga 17220 aaaagtcata aatacatctt tatggacctg gtaagtattcagattagttt atggagttta 17280 aaatggaaaa atgctcccca aacctttctc tgtattcacaacctcatctg tgaagtatgt 17340 tctttcagag ttcaatgcta gagaagagag agtaagtaaaagtcactgag agacattgaa 17400 gagacagtta tgaaaataat taataaactc cctacaggaggaaaacattc atatagtttt 17460 gcggatgaaa ggcatctgtt agatttttgt ggttattttttagaatatac acctttagtt 17520 caagtggtag actattttcc aaaatgtgtg ctgggtagcttaatggaatt atagatatgt 17580 aagggttgtt gggtgtttag ccatgttttc actctctaaccaaattcaac tttagccatc 17640 tcaagaagac agctatttaa gcccttgttt tcaagcagagaaccttactt tccaccaaga 17700 ctaacagagt atattttctc cttataccac agttgaacagggatttgcaa gttgaaagat 17760 ctcctgcctt aggaaacaag ggtctgttat ttctgactgtcttacctatt actccccttt 17820 cttcccatgt ccataatgct gcttagcttc ctggggtgtgaaggataatt ctgtacctca 17880 ggatgactat taggttgatt tggcctgtta ttctaccagacagatcacag agtagagatg 17940 gattttgtgg aacatttgga ttgagagact gactttatccatcacatgaa atttagcctg 18000 ctttggaacc agctgcttag tggggtttag gtactcaggtattcagggaa cctacaccaa 18060 gggttcaaga caggctcttc cccacaaaaa ctatctagatgggagtttga gaaaggtaca 18120 gcaaagacat aacagtggct taaataaaga agaaccataccagtggagac aatgtagtgc 18180 atgacatggc actacatctg ctgttggaag gatcagagataggtgggatg atttgtagaa 18240 ggcttactat aggacaggtt tacagcagat ttttgaaaaaagggtggaag aaaggaacct 18300 aagttggaga tataccagaa gggaagcatg atgtttctcactggaaatta aaaagatttt 18360 aatatagggg acagtaaaca agattgtgga aataatatggagtgttcgag ttatggatta 18420 ctaaagagta agatggttag atgattggaa tgggacagacaggcagatca gagattgtaa 18480 agaacttgga tttgaaaggc ttggtattaa tactgttgctagtttctgag cagaagaatg 18540 acatgaaaat tatgcttagg aaagattatt cttttagtcatatgcttagg ttcagcaagg 18600 aatttccaga ctgtccttag ggagaggaga ggtatggataggatgctggt ccatttagga 18660 tgttagagct gattgaatag gtgctagtct tggggcaagacttagtcata cactttcagt 18720 taagtactat ttcattgcaa ggtctaggtg aagatagggatatatttgta aaactgggga 18780 aattgctaga ttcacagata accaactttg ggcaaatcagttagctttgg tgtccttttc 18840 agtgctatag gggttcagac catttggttt ctgagatcactgctgaaatt ctgtgattgt 18900 atgagtataa tgtaaaagtt gtctcaatca aggaaacttggagtaaagac tcaatatgat 18960 ggatttaaaa ggaatgaatg acagttccaa ttattgggttcaaaaaacat gtgctacaag 19020 tataagatga gaaaaatagg gtacactagt agttcatgtgaaataaggct ctgaatattt 19080 tagctgagtg tgtattccat agcacatagt agtattttgtggcttctaaa aaagtacaca 19140 tatccattgg gtatgagtcg gagcattatt ctttcttgaactgcgctctc acgatgcctc 19200 ctcagtttaa gaacttactc tgaaatagta aaagcatagtgtccagatca ggaggtcatg 19260 gttcagctct gtgctgcact ggtaagaaca tgtgttaagcctttttccat tttgagcctc 19320 actttttaaa gaggtttttg atgaactgga gtcatccagtgatggggagg caggatagcc 19380 tagtgttgaa gcacacgcac tctagagtct acttgggtttgaatcctgcc tcttccatct 19440 actagctgtg aatttttttt tttttttttt tttgagagggagtctcgctc tgtcgcccag 19500 gctggagtgc agtggcgcga tcttggctca ctgcaacctccaacttctgg gttcatgcca 19560 ttctcctgcc tcagcctccc gagtagctgg gactacaggcgcccgccacc acgcctggct 19620 aattttttat atttttagta gagacagggt ttcactgtgttagccaggat ggtctcgatc 19680 tcctgacctg gtgatctgcc tgcctcggtc tcccaaagtgctgagattac aggtgtgagc 19740 caccgcgccc ggcctagctg taaaatctta atctttctaagcttcgagtt cctcatatat 19800 aaaatggaga ttataatata gcactggagc gagtaatacaagagacaata gctatcaaat 19860 gcttagcaaa tatacaatac atagttaagt acttagaaaatatgttttca gttacttcgg 19920 gtatatacct gggaagggag gcagaaaatt ttggtcattctgattattat gactactatt 19980 aagcatagaa gcaccactaa aaaggtgaag aaacctggaaataaccctat actgaggaat 20040 acgccaaacc attgaatcct ggaaaaaaga aaacaaaaggcccacataac tgtcttcaga 20100 ttcttgaagg gctacgtgta agagaagcag aaggcttgttcttttttgtt ctagagggta 20160 gagataacct ggaaattgta gagagacaaa gtttgttcttttcaaagtta agacataatt 20220 tatgtatcat aaaatttacc cttttaaagt gtttaatttagtagacttta gtatttccag 20280 agttatgaga ccaccaccat tatctaattt cgaaacatttttatcaccct gaaagaaatt 20340 ctctactgct tagtagtcac tgtctatttc cctttctttacagctcctgg caacactaat 20400 ctactttcta tctctacggt tttgcatatt ctggacatttcatataaatg gaattatata 20460 atatgtggcc ttttgtgtgt ggcttctttc acttagcatgttttcaaggt tcatgcatgt 20520 tgtagcatgt atcagaatgt cattcctttt catggccacatatttcattt atggatatac 20580 cacattttat ttatctgttc atcagcttat aggcatttgggttgtttcta ctttttagct 20640 attatgaata atgctgctat gaacattttt attatctgggcatatgtttt cagttatctc 20700 gggtatatac ctaggaaggg aggcagaatt tgaagaattaagaaagaaag agctttctca 20760 cagtgggatc cacccacatt gaagactgtt cttttgttgaagtagtcagc cagtaatttc 20820 acttaacaaa tgcatagtgt ttactatgtg ctggatagtgttctaagtgc tttaccctta 20880 tttaaaaatc ctatgaagta ggcactgtta ttacatctcttttacaacag ggaagtttag 20940 taccttgccc aagattacac attgagtaaa tggtggagttgggattgaaa tccaggtggt 21000 ctggttccag agtctgtact cttaagagca gaacaataataactccaagc atttattaaa 21060 ttcttaagat ttatccggca ccatgctaca tgcttgtattcaagcagagg ctggtgatca 21120 tcttttagaa atattttaga aatgattact taattggatagaaattaaat ttcttgaatc 21180 tgaaggttct gttattccaa ggatggctgg aaatggaggaaaagccagca acaagatgat 21240 gtctggcctg aggcttttaa gatgatcaga atttgtatcaaaaaggaaac tgtgaacacg 21300 aatgataagt attataggaa ttcagttaac aagtcttgtcagaatgtggt aataaattat 21360 atacaactta aagagatgaa gttactttaa attgtgtgtggaagccttag aaaaattgaa 21420 taattaaagt ttcttgggaa gatgatctcc tcttacaaatgtttagtttc aaatagtggc 21480 agcacatatt aggatagaga gatgacttct agaaaactgaacatatggga ctggaacagt 21540 gtattttgga gtcagttaca ggaaggaagt tgtttaaaaccaagtgaaca acaacagtaa 21600 caaatgcatt tgagagaaag agcagtgcag tccagacttaaacattaggg tggccataat 21660 agatggggaa gaaggagaaa tcagcaatcc agactaagacttcactgggt tttgagttag 21720 acagagggag tcatagaagc caaggtaaaa tgattaaaacggtgcgtgga gtggttagca 21780 attctcccta taggagagag aatttcctta tgaagtttcagatgagacca ctgaacttga 21840 ctagaggaaa gtcattgttt ttagaaaaca gtttctaagcaataattaga attaaagttt 21900 tgttgcggag ggataagtaa gaagtagatg gaaaagaaagagaaatatct gaaattaggc 21960 tacagtcata gaaggttagc tgtgaaacaa taatttggaaagggaaatgg gacctcatga 22020 aagtgtgggt taaaagagac ttgtatatct tctaaggtaaaagtaaagaa ctaagctgat 22080 gtccagccct ctaaaacatt ttaaatagaa atcaaactttattaatacag taggtctagt 22140 ttcacataaa gcaaatatat ggttctgtac atgggctttaggagagtaag tctgttgtac 22200 ctcatatgta agtattatcc catttagagg aaaaccctaagtctttcttt acagtggctt 22260 aaaggcccca attgatccac atcccagtta cagctctcatctattttctg ctgctccaga 22320 tattcttgct gtttctcaga cacgctaggg ctgctctctgtgttatggcc tttgcatttg 22380 ctgttccctt tctcatgaat gtgtttccct aagatacctgcatgattact ctcaccttat 22440 ttaggtctca actcaagtat tgtcagtgaa ggtcttctctgattttccta ttctaaactg 22500 agatacttct ctttgctcac cccacaccct catccccagctccattcctt cctgtttaat 22560 tttttttcac agcacttaac tccatctatt gtgcatcttttacttagtaa ttttgttttc 22620 gtcttcagct attagaatgt aaactccaag agtgtaagaatttttctagc actggaaaaa 22680 tgcctggcac ataataggca tttactaatt gttgtgtgaatgaatgatta taagtttaat 22740 attctttgtg gaaactaata aggttgattt cctatcagaaaccgatcatt attaataagc 22800 atattggatg gtatttatag aattagcctt catatgttctctactcagaa gttggcaaac 22860 tttttctgta aagtgccaga gagtaattat ttttgcaggctttgtggtct cattacagct 22920 acttgactct gttgttgtag tggaaaatca gccatagacaatctataaat gaatgagcat 22980 ggctgtgttc cagtaagact atttatggac actgaaatttgaattgtata taattttgaa 23040 atgtcagaaa atatccttct tttgttcttt ttttctaatcatttaaaaat gtaaaaaact 23100 attcacaggc caaacaaaaa caggcagtgg tccaaattagacttgcaaac tgtactttgt 23160 caatctctgg ttttactgat ttcttaaata tatcttaggttaaaacttat ttttccatac 23220 ttgataggga gcaacaatgt gaattaaact gtagaaacaaataattcaga aatacatctg 23280 tttagcttta gaaacttcca ttttgcttca tttttgttttcctgggaacc ttatatttcc 23340 attatatgtt tatgatgcta ccatttaaat gactttgcattctcttagca tatgccacat 23400 gatagtgtta gggtggtgta atagttcagg agcaaagctgagcaaagccc acccacgttt 23460 aaaaatttgc cattatttag aaggtgcata attcccatatgggagaatga agagtcatct 23520 aataatgccc agagggctgc actgtgagtg agaattgtgttgcagttttt aacattcata 23580 acacatttct tgaataagct gcaaatgctt ttaataatatatctcagagt taattttagc 23640 tatgctacat tggcactttt tgctcctcca cttcaaaaaaccaacacaag aaactattct 23700 caaatcaagc tgtgatttgg atggggcatt gtattttcctcaataagcac aaagaagaca 23760 gaaaagatgt tttcacttgg caaaactttt ttttttgagatggaatctca ctctgttgcc 23820 caggctggag tgtagtggca caatctcgtt ggctcactgcaacctccgcc tcccgggttc 23880 aagcaattct catgcctcag cctcccgagt agctgggattacaagcgtgc accaccaccc 23940 tggctaattt ttgtattttt agtagagcca gggtttcaccatgttggccg ggctattctc 24000 aaactcctga cctcagatgg tcctcacacc tcagcctcccaaagtgctgg cattacaggt 24060 gtgagccact gtgcccagcc cacttgcaaa actttttaacatttataagg ttttttacat 24120 acttgatttt gttcagttct tagaataatc tcatgatgtaggtgacctta ttaactattt 24180 acaatgtgag aaaatggata gtcagtgaag ataagtaacttgtccaaggt cacagcacca 24240 gaactccaaa ccagaccttc tgaccttctt gatacagactcctcacagtc acctgcatgc 24300 catctttggt ttcagtgcta tgaactttct tcactgcctgttggtctgta gctacaccta 24360 ctcatgaaga tgcccagagc cttagccgct ccttgctccgccatgcttat cttatgcagc 24420 tctgggaggg gtgaagaggc tgtggccagt tacctcactaccactatagc tattcaccac 24480 cctgttttct cctttagcca ttttagctac cgcacaattcttctgggcat tttctgtctt 24540 agttccctcg tatctggcct tccttctttt ctcagtaactgtgacccctg ctagctagcc 24600 ttgccaacca atggttaatg aaaggtcttg gatatgactgtcaaagacaa aagcactcat 24660 gaccccagtg tttcaggtca caatgattag gaaaatcaggacaacaaggc ttgggcacag 24720 aagagctggt ttttgagagg gggatttaga gtttggtttaaatgtgtaat ctttgaggta 24780 atggtggaac agcctactag attcagtttt ctagtccctgaaaactaaaa cgaggaataa 24840 gttgaatcac atatttgggg caccagccaa atatatagcagcatttatag ttagaatcaa 24900 agtgtttgtg tttcttaatt taaatgcttc tcagagattccatttaaaca acttcattat 24960 gaaaatggag acttaccatg cctttttgat attgttcagagaaaattgtc ataaataata 25020 cacacagttt gaaatggttg tacatcttat gaatcaaggggtggcatgta aaaaggttcc 25080 tcagtgcctc aatgtggaaa atatcataga atgttattcttcaattgatt ccttccttca 25140 ctttggtgtt tatatggtat cttagatatt ttatgttattttaattatat tttgtgcctt 25200 tttatttggt taaaaatgcc tactacttaa atattaacagatttttaaat atccctttga 25260 actcttcccc attcctgtac ctcctttccc tttttccgttgtaatgtcag ccacctgaac 25320 ataacccaga aaactaggta tcagccaatt gatgtttcagtggaaataag agaaagaagc 25380 gcttactatt tctcaatgct taattgagga aaagcacatgatatcttaca tattttaaat 25440 tggaggggat aatagggtca agttcatgca ttatattgtggacatcttaa tcctatcaaa 25500 tggcttagtc aaaatgagaa agttaatgaa tttaagcagacaagtacgaa cagtgctcta 25560 aaaaaggtgc tgttacacca tcccaacctg tacacacatgaaaaaaagct ggggtgcatt 25620 ttgcgtttga ataagggtca tgttgactgg ccatggcagaccctgctaga ccatacacct 25680 gatactctgt cacctgccag gactcaaaag gacatgggagtatgtggtta agttctctaa 25740 ggactctttc ttctaaaggg aagcagtttt ataggtggtccttgtaggtc tgttgattca 25800 caatctcgtg ctttcttgat tggactgtat tgttttactgtaattttttg gacttacaag 25860 aagtcaggtg ttttcatgga ctcttctctt ttccatacagatgtccagaa ggcttcttgg 25920 gggaatattg tcaacatcga gacccctgtg agaagaaccgctgccagaat ggtgggactt 25980 gtgtggccca ggccatgctg gggaaagcca cgtgccgatgtgcctcaggg tttacaggag 26040 aggactgcca gtactcaaca tctcatccat gctttgtgtctcgaccctgc ctgaatggcg 26100 gcacatgcca tatgctcagc cgggatacct atgagtgcacctgtcaagtc gggtttacag 26160 gtaactaatg agaccaaagc cagtggtttc ctaccttcagcagatacctt tatttagcat 26220 cttttagatc atggtgtctg gctcttaaat gtcccccagctctggtgcac atttaacatt 26280 atgataagga actgggatgt tccagacaac tatccctaacttccttttaa gagtttcagg 26340 gggcagagaa agagaaagaa aaagtaccaa atactttgactgcttaaagt atatatgtca 26400 gggccaggtg cggtggtgca cgcttgcaat cccagcactttggcaggcca aggcaggagg 26460 atcacttgag gctagaggtt tgagaccagc ctgggaaacacagcaagacc ccatctctac 26520 aaaaaaacaa gaataaaaat aaaacaaaat tagtcatgtgtggtggtgtg cacctgtagt 26580 cctaactact tgggaggctg aagtggaaga attgcttgagcccaggagtt tgaggctgca 26640 gtgagctatg atcgcaccac tgcattctag cctgggtgacagagtgagac cctgtctcaa 26700 aaaaaaaata tgtacaccag gatggggaat cagagtttacttcactaaaa gaaataagta 26760 cactgtcacc agaggaaaag ttgctgaagt tattgactatttgcttttag aaatctccct 26820 ccctagacat tcagggcact ggcttttctg gttttctgaaccctgttcct tttgcttctt 26880 cattaccttg ttcttatcca ttaaatgttt gtgctccctggagcactgtt tttcgccctc 26940 ttttgagcca catcacagct ctccctaggg aatttcactgtctgtattcg cctccactgc 27000 cactgtcttc attagcttgc tgatgaatct caccatcattcccttagctc cacccaaccc 27060 tgacattcag gctcatgttt ctagctatcc tttgtatgttctccttggag atattctaca 27120 ggtactttca gctcaccttg ttgacagaaa tacgtagcaaccatgtacat cccaaatacc 27180 cacgctagaa actccctgtc ccttgttctg acctcatttcaactcagtca cccaagccaa 27240 cctctgagtt gcctttcacc tgtctattcc tcccatttcctctgctaccc tgtagttgag 27300 ggccatgtta tctctcacct ggacttctga aatagtttctgaatacgttt tcttgccttt 27360 attctctccc catctcattc acccatgata ttactacatctttgattata aatgcaaata 27420 ttctaacaat ctcacctgct tacaatgtct aatattttttcatcatccgc agaataaact 27480 gcaaactctt ttacatgact tccatagctc tctacagcctagactttaca tctttttgta 27540 gcctggcctc cccacaagca tctaggccta gtcacaccaaattctcctta tttcctgaaa 27600 atgttgtact tattattgct tccatacagt tacacacccttttgcctgga atgccctttt 27660 ctacaactgg tgattgtcca atgttattta aaactgtgtcttagtgaccc tttcatgatt 27720 cctttaggca aatggtctct aagttttatt agttttatgtatcacatttt attgtaattt 27780 ttttttacac atatctcacc tgaatagatt gtgggtttttctaggtgggt ctgagcttta 27840 ttcaaaagtg tttattaaat tagatgagaa aaggaggaacattcttcatt ttttctcctg 27900 ctttaagcac taaaccaaga gttctataaa tgcaataagcaaaaaagtga aaaatgtact 27960 cagaaaacta tactggatca gttagtgtag aatactgttatattaatttt tcattgtatt 28020 agggttctct agagggatgg aactaatgga atatatatgtatgtatatat attccaaccc 28080 aaagtgtctt ggtggcaatc ttaatatata tatattggcaatctttatat atatataaag 28140 gagagtttgt taagtattaa ttcacatgat cacaaggtcccacaataggc tgtctgcagg 28200 ctgaggagca aggagagcca gtctgagttc caaaaccaaagaacttgggg ttcgatattt 28260 gagggcagga aacatccagc acgggaaaaa gatgtaggctgggaggctag gccagtctcg 28320 ctttttcatg tttttctgcc tgctttatat ttactgacagatgatcaaat ggtgtccatc 28380 cagattaagg gtgggtctgc cttccccagc ccactgactcaaatgttaat ctcctttggc 28440 aacaccttca cagacacacc caagatcaat actttgtatccttcaattca atcaagttga 28500 cacactcagt tttaaccacc acaaatctac cccttgtcaacttgaaccca tacacatctc 28560 ctgagatcac acataatctt caaataaaga caataattaggtcataatta cacctgatgt 28620 agtacaacta ttcttcatac atccggaaac acaccagtccccagctgaaa cactcttaca 28680 taaagttaac gatacttaaa tgctgatgtg aagtcaataaatcttatgtc acatgataaa 28740 ggagaaagga aataaaatga agatattttc ttagtacaagtgtgtgcaag cacagacatg 28800 tttttaataa aagaaggagg aaatactgag gacaattacagtcctcattt ctgcagctgg 28860 tcacatggta gtagctggta ttgatgacta ccttcttctacccattttgt attccttttg 28920 ccttcagcaa acacctcagc aggttatgtt tttttttttcctgatggaga ggcccaaacc 28980 ttcattcatc caggtgggac catttgtagt tccgcctggattgggctgtt gtagttgccc 29040 attgacctta atcacagggc atggtaatac taagagacgccctaatggat ctcctgtatt 29100 ccatgaatac tctttcttac ctccgttctg gagtagtagactgatttcat cttgatagcc 29160 tgggtcttga tagccttgat gtcccagcca acactgtaacgcctttctta ccctgtttac 29220 ttaaaggtag gaacccaaag tgtcctggtg gcaatcttaactaccagttt aatggaattg 29280 ttgttgtgtc ttctggtggc agcattcctc cctctggaactaagacctct aggccagcag 29340 aacttaatgt tgcgggaaca ggaagcaaac attttgctagtggatcacta ggggtgatgg 29400 tgagtggtac cacttccatt tccacccctt gattcctggacctgtgaatc ctggctatgg 29460 gagaagcagt accacatatt ggacgctgat tcagagcatacacagccttc ttgagaactt 29520 tgccccaacc ctgcaaagta ttctcaccta gttggcattgtaattgtgac tgcaaaaggc 29580 cattccaccg ttctgcttca ggatgttggg gaacatggtaagaccagtga attccatgag 29640 catgagccca ctgctgcact tctttagcca taaagtgaatgccttggtta gaggcaatgc 29700 tgtgtggaat accatgacag tggataaggc attccatgagtccgcggatg gtagtcttgg 29760 cagaaccatt gcatgcagga taggcaaacc catatccagagtaagtgtct attccagtaa 29820 ggacaaacat ctgccctttt cacaatggaa gaagtccaacataatcaacc tgccaccaag 29880 tagctggctg atcaccctga ggaatagtgc catatcaaaggtgcagtgtt ggtttctgct 29940 gctggcaaat tgggcactct gtagtggcag tagccaggtcagctttccac cggcccatct 30000 tgttttctgc actgggaagt tggggcatga gcacaaatgttaggacctga aaggtggtga 30060 actctgcctg ggcagggcaa agccagagga aactctggtggaggtccgta gcagtcctga 30120 tgtgcaaatc ggtcatccaa cctgggtgta ggggcgaaagactaattgaa ccatctagta 30180 gctggttccc tccaaagttt ccctcgggat agttggcactctggcaaaaa ccccactctt 30240 ggtaccaatt tactgtatta gtccattttc acgctgctgataaagatatc cccgagaccg 30300 gaaagaaaag aggtttaatt ggacttacag ttccatatggctggggaggc ctccgaatta 30360 tggtgggagg caaaaggcac ttctttcgtg gtggtggcaagagaaaatta ggaagaagca 30420 aaagcagaaa ccctgagaaa cccatcagat ctcatgagacttattcacta tcatgagaat 30480 agcacaggaa agactggccc ccatgataca gttaccccactgggtccctc ccacaacaca 30540 tgggaattct gggagataca attcaagttg agatttgggtggggacacta ccaaaccata 30600 tcattcatca aaaggtgttg gtggggtaga gggtagttaggatgatccat tatctccatc 30660 atgacgatga tggtattgat gatgtaagtc actgaaaatatttggtgtta taagaataat 30720 ttcttcctga ttgtcagttt tgagttgttt tgctattaggaggcaaagta ggggggcata 30780 tacccactta aaatatttta attctggcta gtggaaatgacagtaatgcc ttcttcataa 30840 ttaaaatgtc actctgaaat ggtcccaaat taaaacttcttcctttgttg ttaagaagga 30900 tctcttcttg gtgtgttctg caagattctg atcacctttttttttttttt ttttttgaga 30960 tggagtattg ctttgtcgtc caggctggag tgcagggcacaatctcggtt cactgcaagc 31020 tccacctccc gggttcacgc cattcttctg cctcagcctcctgagtagct gggactacag 31080 gcacctaccg ccatgcccgg ctaatttttg tatttttagtagagatgggc tttcaccttg 31140 ttagccacaa tggtctggat cgcctgacct catgatccacctgcctcggc ctcccaaagt 31200 gctgggatta caggtgtgag ccaccacacc cggccgattctgatcatctt ttatacatat 31260 gctatttttg tctatcactt taggaatcat cacagatcaaagtcatcctt ttggtttttg 31320 tgatagcact atacctcagt cagcttacta gctcatctccactcagagat gaagaagcag 31380 aggcagcaag ttagtgccta tacataatat atatggaaaccaaattcagg gttgattctt 31440 tctttctttc tcccttcctt ccttcctttc tcccttccttccttccttcc ttccttcctt 31500 ccttccttcc tttctttctt tctttctttc tttctttctttctttctttc tttctttctt 31560 tctttctttc tctctctttc tctctttctc tctctttccctctctctttc tgtctgtctt 31620 tcttctcact ctgttgctta gtacagtggc gcagtctcggctcactgcaa cctccacctc 31680 ttgggttcga gtgattcttg tgcctcagcc tcccaggtagctgggattac aggcatgcgc 31740 cataaagccc ggctaatttt tgtattttta gaaaagatggcatttcacca tgttggccag 31800 gctggtctca aactcctaac cacaagtgat ctgcccacctcagcctccca aagtgctggg 31860 attacaggca tcagccacca cttccggccc agggatctttctgtttcagt tgtgggcatc 31920 actctgaaaa tcacacttgc tagaagtgag catttatatctcttctccac tgtaaataag 31980 tgcctcttag tgacatgagt ggaaagacaa gaagaattgcagttccttca ttttctgtct 32040 tagctccctg agatgtatat gctgtgccta aatttgtgttatagttttct cctttgattt 32100 gacattcctt gataggcaga gagcactttt ctgtgctcatatgtcacatc tcgcatcttt 32160 ttcccttata gaaaaactct tgtgtctccc atttacctttctatgaggtc agagatttag 32220 atactttcct agacaatcaa ctggagtatt aacaaattcaaggagttctc gccatcccgt 32280 tactggatat atacccaaag aattataaat cgtgctgctataaagacaca tgcacacgta 32340 tgtttattgc ggcactattc acaatagcaa agacttggaaccaacccaaa tgtccatcaa 32400 tgatagactg gattaagaaa atgtggcaca tatacaccatggaatactat tcagccataa 32460 aaaaggatga gttcatgtcc tttgtaggga catggatgaagctggaaacc atcattctca 32520 gcaaactatt gcaaggacaa aaaaccaaac accgcatgttctcactcata ggtgggaatt 32580 gaacaatgag aacacttgga cacaggaagg ggaacatcacaaaccggggc ctgtcgtggg 32640 gtggtgggac gggggaggga tagcattagg agatgtacctaatgtaaatg acgagttaat 32700 gggtgcagca caccaacatg gcacatatat acatatgtaacaaacctgca tgttgtgcac 32760 gtgtacccta gaacttaaag tataataata ataataataataaaaattca aggagttctc 32820 atctctgtag tttaaataat aagtgactta gactaatgacaacaaaaagc cagccatgtg 32880 aataccaaat ttactagttc tgtgaggata tttttttctctttctctttc tgcctcaaag 32940 aatctgcttt gcttcccctg ccatcatgat ttagttttcaacccgtcaga gtcttcctgc 33000 tagtgctggt actttcctac ttgagaaagt ccacggaataccttcgagac ctctgtcctc 33060 ctgatggctt ctatttcatt tgttatatag ggacccagagttccttcatc attttcaaac 33120 acatcaacag atatttatag caaggccaca attaataaaatgtttcccag aatatatgtg 33180 tgtgttacat ttagaggaaa cagaagtagt attgacttgtttctatcacc agaggtctat 33240 ttagtaacta tattttgtgg aaaatatcga tatattttatccattcaaca gacatgattt 33300 gagagcatac catggagacc caaccctgcc agtgtggcaggtggtataat agaagaaaat 33360 agcaaacttg gtgtatctgt gtttgcgcac atgtatgtatgtgaggggca ctaaggatga 33420 ctttacagag gttggaactt ttgagtacag ttgccaagatagggagagtt cactaggaaa 33480 acagaaggga agttgatttt tttttttttt tttttgaaatagagtcttgc tgtgtcgccc 33540 aggctggagt gcagtggtac aatctcggct cactgcaacctccgcctcct gggttcaagt 33600 gattcttctg cctcagcctt ccaagtagct gggatcacaggtgcgcgcca ccatgcccag 33660 ctaatttttg tatttttagt agagacaggg tttcaccatattggccgggc tggtcttgaa 33720 ctccttacct catgatctgc ctgcctcggc ctcccaaagtgctggaatta caggtgtgag 33780 ccactgctcc tggcccggaa gttgatattc aaacaggagcagcatatgca aagacagtga 33840 gctctgagag agtagatgga tccagactcc tattgctgatagcgtcctgc aggattgggc 33900 ttcaatgtga ctaacctaca attgcctcca ggtgctccacccactgagtc cttgtgtctc 33960 tgctgaggtc cttggagagt tactggagag ggctctgtgtcagattacct tgaggaggct 34020 ctgatttagc cttttgtaaa atgcaaagag ttgaggtcttctccacgcaa gagctcgctg 34080 atgtcaatga ggtattgagg atggggccat ctcctatttctgtggccagt actgagtttt 34140 gttatccttc ctttaggtaa ggagtgccaa tggacggatgcctgcctgtc tcatccctgt 34200 gcaaatggaa gtacctgtac cactgtggcc aaccagttctcctgcaaatg cctcacaggc 34260 ttcacagggc agaaatgtga gactgatgtc aatgagtgtgacattccagg acactgccag 34320 catggtggca cctgcctcaa cctgcctggt tcctaccagtgccagtgccc tcagggcttc 34380 acaggccagt actgtgacag cctgtatgtg ccctgtgcaccctcaccttg tgtcaatgga 34440 ggcacctgtc ggcagactgg tgacttcact tttgagtgcaactgccttcc aggtaaggag 34500 ctccctagtg tcccaggatt aggggacaaa cccctagcacaggaggtagt gggtgtggct 34560 caattgctgt ttttaggaag cccaaggaaa aagggaagtgagaattttgt gtggggtggg 34620 ttgctagtga gggaggagtt ttatgggccc actgtggtccataaactgag caggggataa 34680 tttagcatgt cagggtttat gatgatgagt ggctagaaaattgtttattg tcccttttgt 34740 agaaacagtg agaaataaga ggaacagagc tctgggaaagggacaggcaa gtctggaatg 34800 gaaaagaaca cgatgagaat tagacactgg aaaatatgtatgtgtggtta ataaagtgct 34860 ttaaactgaa ttgacattaa cagtaggtga tcaactttcctatgtgcttg tgcttttgct 34920 tttgatggag taattcattg ttttcttatc cacctaaatgcacccagctg cccttgattt 34980 tctctgggct actggccttc acaaccctct cccatgtaccctctctgact ttggggtaac 35040 cctcccctaa cttaaagcta gagaattctg aaactgaggaggggatcctc tgttaatcag 35100 tgagcacttt ttgatgagct gatagatgat atatgagagactatgcgtgg cacaatactt 35160 tgttacactc ttcactgata caagtgttct agagtgcacacacaacccaa agatagaaac 35220 aaaaagagga gcagtgtcgg ggagcttggg gcctggtgttccatggagag ggagaaagga 35280 acaagcctgg ccaattcatt caactcctta taaaaatgatgaggaggctg aaaaccaaga 35340 attttgattg ggaacagaat acaagcagct ggagcagatgaattactaag caacaaagat 35400 cctgttttta tacaaatatc cttagtacaa aaacaaaagaaggaaaactg taggggggag 35460 taatgtgcta agtaagcaga attgcctcaa aaagaagttgttctagttac tctttcagag 35520 tgggaatctt agattctggt attgtggata tggttcacatataatgggat tgtgtgtttt 35580 attttggaga gattaaaggt cataggttgg tcctcagtataaaatcaact ggtaatttat 35640 tcatttcatt tggtaaaaat gtattgactg cctgctatgttctaggcacc atgctatgta 35700 tttggaatac agctatacaa agcattgtca cataattgaaatgaaaactt tatattattt 35760 aagtcacaag aacaagctat ttaattatat tacttttagtttctctttta ataaagaata 35820 gataatgcta tcattctaga tactaaataa gtgttttcttaacataatat tactattcac 35880 tttatcttgt agaagaaata actaaaatac gtctgtcttcactcctgcat ttgtttgcat 35940 tttaagggtt aaagacagaa atagaaatgt aaacaactttattttgaaaa tatttcaata 36000 ttgcaaatat ctctgggtct gatattctag taatctaattggctagtaat tgatgttagt 36060 gtgatttatt gttgaaggct aaatgtgttt ttcagtttcaagaaaattgc ttttaataat 36120 tgcctagaac aagaggttga tttggcagca agatgttgacgggaagttag agaagtcaat 36180 aaaggaagtt tttagctgag agagagtgat tattcactcccatagcctct gcattgttat 36240 ccattagcca cgataagaac cttagggaat tctgagagtgtgtccaggaa aggatctgtc 36300 aaactagaat agtatctcct ccttgagaaa ggaaataaccaaggattccg cagctgagag 36360 gctgccaggg ctagtgaaat agagtaagga aatcttggctgtctcttatt ctctggttgt 36420 agtttaacgc aagacactta tttactcact gatggtgtgtgtgtatgtgg gaggggagat 36480 tagcatgagg ggtgggaatg gggagatttg atgaagagaaaactaacatt tttttggtga 36540 ctcaggaact gtgccaggta cttccatgct tattagctcaattacacaaa aatcttggga 36600 ccaggtatta tttttcaagt cttcccacat gaagtaactgaagtttggag atgttaagtg 36660 attcacccaa agttgtacag ctaatatgtg gttaggctgggtcctgaaac cgaggcagtt 36720 tattttcaaa gcctttgctt tgtgcatctt actgcgccacattgcactgc acatctgctt 36780 cctgaaagca ctttgtaggt gtgtaaaact tttcgttaaatgctttaagc tgtttgggtt 36840 aaaaatatat gttcatgtta taagaaaacc aagacactcctaattataat caaatagtac 36900 ttgttacata tcaatatgtg tgtgtgtgtg tgtgtgtgtgtgtgtgtgtg tgtgtgtgta 36960 tatatatatg gcattgtgca gatgtttaaa agtagttacatagactagtt cttgcttttc 37020 agggtcccat aatctaaacc agatgacttc agctttggataaatatatag aaggaaattt 37080 aaagagaatt caaaacaata gatgatgcag tgacactgtgaataaatgtt atttatacag 37140 tttgtaagat ttcatgctca ttgttcgtat gtcccaggtggagttcagaa aatattcact 37200 tcatttccac aaagggaaat agtgcctaga gatggttttcttttaaaaag tccttttcat 37260 aatgcagtgc ccttccttcc attgcccttc attccattgcttcccatgct tgtcaagaga 37320 ctaaaatgtt acttatagta atagtcacta tctcaatgtaaatagcaccc ttatttgatg 37380 agaattatta tttcagttct aaaaatgggg aaacaaagtcagcaggaggc aaagtagctt 37440 gttaaagtat tctgcaactt tcaaatggtt gcttccactgcatttcacgt cttggcactt 37500 ctaattgagg gttactctaa ccaccctatt taaaattgtaactgtccccc acccccttaa 37560 ttactaaccc tggtctactt tttgttttct ttttctgtaactcttatctt cttactatat 37620 aatttatact atataattta cttcattatg tctattgtttattgtctgtc ttttccaaat 37680 tctactgcct cttaccctct ccagaatgaa aactagtatctttgtttttg tttactgatg 37740 taacccaaac acctacaaac agtgcccagt atatactaggcccgcaaata tatattggct 37800 gactgactgt atggtttagt atcatgtcat agtattgagactgtaacttt ggtcttctca 37860 ttttcttctt tgtattgtgc gtcctagact tagtttggcctctccttttg tccttgtata 37920 ctctaatact ggataagaat tttggagtct ttttcaactctgagtcagtg aatgccacat 37980 aacttagtga ctatatttaa atggttaatt tacaattttttccctgcaaa ggatactgta 38040 gtcactgtga gtattttagt attattgtag gactcaagagggaattaaaa ctacaaaaat 38100 gactcgtctt gtatgacaca gaaagaaatg tttcttcacagagggaggag aaaaatatct 38160 tcaagagaga actaattgaa tcaaatcaat gaaccatgtctcatcttttt ggataagtaa 38220 ctgttagtaa tccagacact tcatgagctt tcattatgtaaagtctttag cagaagctaa 38280 aggaggggca ccaaccacag taattttaac ttaagaacaaaatggagcat gaaaataaat 38340 tattaaatca tttactccca ctatttttgg gttagggccaataatgggga gagaaagagg 38400 tagactagtt ttgtgtttgt ggctatttta atagagtagcacaagtaatc aaaaaacagt 38460 aggctgtttt gaatttactg gctgtccctt atgagttcacagttagattg gactgtcctc 38520 aatgtacttt cttttttttc tttctttccc acatctctttatttctctga ttttgtttaa 38580 acttcataaa gagctctctg atctttcctt tccaaacaatgaaggtttat cctttgtaaa 38640 ctacctctgt actccacagg ctgatgatat atgatatccctatatcatta aagtaaagcc 38700 taagcacatt ctgtggcttt tgtgtctact ctgttgttgctgagcttatg aactattaga 38760 aataattccc tcttgcattt tcacacatgg ggaatgtgatgttctcttgg gtattatgct 38820 aatcatattt tggcaggttt ctctgaagca gatgcagaaatgatcatacc actttccagg 38880 gtgtattatt ttagctcctt tgacttgggc cctaagtctgttttacctga tgttcctgaa 38940 agatgttcct gatgtccctc actgttcttt catgctggatgttcttgcct atgctgcctc 39000 ctcagctatc accctctctt ccccttttta atgtagaactcattcttaat gatttgtcaa 39060 aggcacccta tttcactgaa atgccttcta tattccctaccctccaagtg gattgtagac 39120 cttctaaggt cttttgacat ctgcatatct ctagcacagcacttatcacg gtgattattt 39180 atctgttcat ctttccaagt agacactctc attttaactccctaccctag tcgccagcat 39240 ccccagcata gtgcctgtca taaaatggtg ccacaatgaaaatttgaaaa atgaatgaat 39300 tgaacgtgat aaacatagat gagaatccta tattctacaattttttaaat gtactgaaat 39360 tattcttttt gaatcctcct atttatttct gtgacttctttggtgacaaa gttagaaaaa 39420 agtggaggtc agtagggaga tatgaaggga cgcaggtggaagcagtgagc ctgggcgggt 39480 gatggagtgg gcgatacgtg gcacaggggt cagtgagttaatctgggctc attcagagaa 39540 tggaagttgt gtgccaagaa aactggttgg atagggataggtcagggatt ccctcttgca 39600 ttctcacact tgggggcatg cgtcattttc ttttcttttcttttcttttt tttttttttt 39660 ttgagacgga gcatcgctct ttctcccagg ctggagtgcaatggcgctat ctcggctcac 39720 tgcaacctcc acctcccggg ttcaagctat tctcatgtctcagccttcca agtagctggg 39780 actacaggtg cctgccacca tgctcagcta atttttgtatttttagtaga gatggggttt 39840 caccatgttg gtcaggttgg cctcgaactc ctgatctcaggtgatccacc tgcctcggct 39900 tctcaaagtg ctgggattcc aggcatgagc caccatgcctggccgcatgt gtcattttct 39960 tgggtgttat actgatcgta tatttgcagg tttgcttttgtgacagactt cttctggggg 40020 aaaaaaagta tccttctatc tttttacttt tgtccagttccaggtatccc tgtttttttc 40080 ttcactcttc cttccttgtt catgggagtt tttcttgaggacttcaagcc cagcttcgga 40140 gaatcctggt tgtgtcatct catctccttt ctgctctcttctctacctag gctttccacc 40200 ctcacacctc ccggggtctg aaaatggaaa gataagggtgtttccctgaa agttgctctt 40260 ctgtgtgggg atgacaggtt ctaaagactc ttttctggtccctgccctca ttgccatgat 40320 taatcagtta agtggcccga ggttttgtaa cagcacagtcttaaaatgct tctcccaagt 40380 ttaatttctc tccatttgac cttttaagga tgtgaattggctttaagcag tagactccct 40440 ttagtacggc actgtgagcc tctcagtgaa tctgctacatccattccacc cacgggtctg 40500 gaaacttgtc tgtttacctt tccctaaaaa cctaagatatatttttaaga agtgccttgt 40560 aacttttcat atagcctttc ccctactttg ggtagactgtttcttacagg aatttggtag 40620 atctttccaa agagaattct gtatctctat ttttaaagcataaatcctgt caactttgga 40680 ggagaactga tttggcttga gtcttctcag acatgggaacttttgaccta agtttgtatt 40740 ttacattgtt gaaagggaac tccgggatcc cagaaaacatatggactgca attgggtaaa 40800 gtttctgttt cagtacttat tcctacttac tagccgtttaatcttggtca agtcagccat 40860 gtggctctca acttcctcat ctgtaacata aaaggattagagtagacaat ctctaacaag 40920 tgctataata ccactgacaa ataataatat tagctaatatgtgtaaggca ctgtgtttag 40980 tgctttttcc cttaatacaa tagctttgag atataatttatataccatac aatttacttc 41040 ttaaaaaagt acacaattca gtaattttag tagataggagtaaccatcac cacagtcaat 41100 tctagaatat ttttatacat cagaagaaac cctttacccattatcaatta ctctccattc 41160 ctcctaactc cctcccagcc ctaggcaact actagtctactttctgtctt tatttgcctc 41220 ttctggacat ttcatacaaa tggaaacatg cagcatgtagtaatttatga cagcttcttt 41280 cacttagcat gaggttttca aagttcattg atgtggtagcatttatcagt actctgtgcc 41340 tttttatggc tgaataatat tttatcatat ggatttaccacattttatca ttttatttat 41400 ccatcatcag ttgattgaca tttgagttgc ttctactttttgagtattat caataattct 41460 gttatgaaca ttcttgtata attttttggt agacatttatcttcatattt cttggatata 41520 tacctaggag cagaattgct gcgtcagatg gtaatgctgtttaacctttt caggaactgt 41580 cagactgttc tgaagtgggt acattatttt acattccaaccagcagtgta tgagaattcc 41640 agtttctcca catcctcatc aacagttgtt attgtctgtcttttttatta tattcgtctg 41700 taatgtgaag tgtttatctc attgtggttt tgatttacatttccctgatg gttgatgatt 41760 ttcaacatct tttcatatac ttattagtca ttatgtatcttctttggaga atgtctgttc 41820 agatccttta cctactttat aattggttta tctttttaatattgaactgt aatagttttt 41880 aaaaaatata tcctaaatac aagtctctta tcagataatatgatttgcag atattttctg 41940 tcattctata tactgtcttt tcacattctt gatgatatacttttcagccc aaatgttttt 42000 aacttgatgg aatacaattt attttttctt ttgttgcttgtgctttcagt catatttgtg 42060 aaaactttgc ttatcccaca ttacaaagat ttactatttctaagtgattt ataattttac 42120 cacctacctt taggtctccg atccattttg agttaatttttatgtgcgag gagggagtct 42180 aacttgattc ttttacatgt ggatatttag ttgtcccaggaccatttgtt gaattaagtg 42240 ctgtttttat ctaattcgtt taatctttac aataaccatttaaggtgggt cctgttaatt 42300 ccccaagaaa gtttaagtat attgcccata ttcactcagctattgcatgg caatgctgag 42360 agttgagcct agacagtttg gccccagaag ccatgcttttagctatgaaa taactgtctt 42420 acctacctcc cttcttacct acctatctac ctattgacagggaaaaagta cctcaacaat 42480 agtcaatcag ttataaaaag aaaacacttt atttcttttctgttgtggtt ctgctgaaat 42540 gcttctgctt tcttgtctgt ggttggtgaa ttcagttagttgtaacattg gctaatataa 42600 agttagagtc acagtttcta ttccttatag ttgagctacaagggcttcgc tacctccctg 42660 acaccctagt caactgtatt tagtcttact catcagagagatcgagttag gggaggatga 42720 tcattgacct tctactacac caccagacct ctcaagtttagcagttgtag gccaggggcc 42780 ccattttccc caacaatcta aaactatgtc tttaaattttccaaagaata tcttcatcaa 42840 gagcaccaga ctaggggcaa agaccctagt tcttactcaaggacctagta gatcccatag 42900 attctgtagg ggtgggcttc aatggctcat cattttctggctcattggtc tatttgtaag 42960 actcatattt ctgtatgaat aatatgaaat atgagtaatatgatacattt ttgaaatttg 43020 agtaaatttt catagtcttt ttcaggcttc ttcagtgattcatctgcttg aatggactga 43080 gtaccaaata cttggagaaa tataatttcc ttcaataaagccttcagtga tttctgtctt 43140 ccatggagtg atttttggct aatgtttttg gtcactcagtttatacttat ttatatgact 43200 gccattgtat ttagagaaat cagccatgtt ccagtgtagcagagcagatg tagaatgcca 43260 cagtaactta tggtaaggaa taaagcaaag tgtgattgatgtgtgtccca ctcacaaggc 43320 tgtaaatgcc acaaaagcag ggactcactg ctgtatcaccagtgcctaat gtaagtccca 43380 gtaagtattt tgagtgagag agaggaacct taagggatatttcgaggaag ccaaatatgt 43440 aatcatacta tatttcaaat aggagaatgt ttagaatagaagctggtagt cttctattta 43500 attagctcag gagtttggta gcaaggaaag agaaagtaagaggcccaaca tataaactac 43560 aaaggaccac actgtgggca gttagagtga agtttggtgtactagtttca tttttcataa 43620 aagaaaaagt caagaggaag gaagcaatgc tgtaagtttagagaagagcc tgatgaattt 43680 gaagtgctgg taagcttagt tagtaataat gccacccaattttcagatac cttttaacat 43740 ttgtatagca ctttactcat gatgtggcac ttataattagaaagacagga taggcaagtg 43800 gaattagcct cattttttac aggtgaggaa gttgatgcagaactttagct taccatgtac 43860 gaatagcttg caggtggtga gattgggact tgatccatatcttcatactt taagttcaat 43920 atttttccta tatcctcccc cactcccttc cccacctggcttggaggccg cctagcaatc 43980 attcaaagaa aaaaaataaa aggaacttaa atttaattaggtaatatttt atctgttctt 44040 cccattagtc tggtatggag ctggggaaaa tccaataccctgttttttta gaacccttga 44100 atatctctgt cccaagtaga catttatcat ccctgggattttatttcatg aaaacatttt 44160 gaaatacttg tgaggtaact atgaaggcat gccattggcacaatgagtca ctttcactgt 44220 ctagctatgc ttgaggccag agaagacaga gaattggggatttttcatat ggtgagtcct 44280 gggtgagcag ctgttgaact gaagtcatga agcgggtctattctaataac catgttctgt 44340 gggaacagag caggtcgttt cttctgtatg agatttcatcattttcattt gttcttcatt 44400 taccaggttt tgaagggagc acctgtgaga ggaatattgatgactgccct aaccacaggt 44460 gtcagaatgg aggggtttgt gtggatgggg tcaacacttacaactgccgc tgtcccccac 44520 aatggacagg tatgtacagt gtggagaatc caccagaatgggatatggat tggtaaccag 44580 aggcagactg tttttttaaa atatcagtaa caaatatcttaggcctgctc tggaaccagt 44640 atgtgaactg agcaggatct ccatgctaga tcattgcttattgtactaac ctcatacatt 44700 ttcctatgtt cactcaaaga catattttca caggtattattgggcagcaa ctctgaaccc 44760 agcaccatgc tagatgctgt ggcagatatg agagaagagcactactgtaa gcttattatc 44820 tgggaaaagc aggacattac ctggaacata gtagatgcttaataaatatt aaagaggaaa 44880 aatttcctga aatgctaaaa taaattgtac aggctaccagagttcagagc ctatttgttc 44940 acggacagag atctgtccca tcttaatgtc atgttctataactaggcaaa gtgttaggca 45000 ggtactctta atgacattat ttttatgata cgcatcctacatttgttttt ttcttctatc 45060 cctttttgta cacgaacaaa aatagttttt tctgcctctaacattagaag ggtagtcttg 45120 cttaatttta cagcctcttg gaagtttctc atgtagtcttaggcaggttg agtgatttac 45180 gcagacattt ggttctaatt tctctttatg tctgtttatctactgaaagt agagacaatt 45240 aagaactgat taagggacta agagctgggc ctttttgagagaaattaatt taatgtggaa 45300 acccaacagg aagttagtat tataagaaaa aattacagtaccagtagtgg gctatctcct 45360 acaaagaatc ttaatgttga agtgcagcca ttactacctggtattcttta gtgtctctga 45420 attacttttc cttatctgta gcatctaatt atttacaatactctttagag ctgtagacta 45480 tgaatagaaa aagatgaaaa gcatttaaaa actcaatcacaggacttact ggggaggcca 45540 aacatttctg cttttaattc ccaaactgtc ccttttaatactttcccacc aggttttcct 45600 agaattggga cttctcctag aatttcttca tggtgtcttgatcctagaac ttcacttcta 45660 accctctgta ctttttgtga cattgtctct tctagtttttgccatttttc tatgatagac 45720 ttttcagagg ctattttttc aacttaaaaa attactgtaaagttaattac atgacatata 45780 gtaactggaa aaatttgtgt tttttttctc tttttaatgatccttacagt attggacagg 45840 atgagatact tgttaagaac ttgaccacat tctgatctctcccttgctct gtgagacagt 45900 aactgtgtag tactgaggct tctgatgcca gtgaagattaaacaacattg taatattgtt 45960 gctggttttt gttcgatact acacttggtt tcttgctttgatattaatta ggaaggcctg 46020 ctcttgggga aaacttcccc agtgaaatga tttttttattcaaatttctt atcccaggac 46080 atttaataaa tgcctctttt cgctagaagt ctttataagatgtttccgtt tttatatttt 46140 ccaaaaaggg tggtggagat catggataag tttaatgtggcagttttatt tcttttgatt 46200 tgctgtctgt catttagagt gaccttcagt tttttttttgttattcatca caggttttct 46260 tcactcatgc caattctttg attcccagtg agaagccttgtttttgccct gcccatcatc 46320 aacaggagaa agtctaagat ccttcttctg ttatatgaagcctttcatgg cttcagctct 46380 gtctgcttcc ccagtctcct tctcatgcat ctagcagcataccctagcag ttctctgctc 46440 acagttttcc tgtacatact gcatagtgct tcacatcctgtgtcttcatt gacaccattc 46500 cttttctcta gggtttcctt ctccactcct cccccatttttctcatcatt tttggcatgc 46560 ctgactcaga tcaagtggtt acctgcttca ggaaggcttccctgatcttg ctccaggctg 46620 agctaaatgt tctactctgg actcccttag taccccatgtattctcctgt aataacactt 46680 accacgtttg tattgatatt attatatgct tgatgtgagggatgtaccga gttatggggg 46740 cggttggact attgatgaca gcagttacta gcactccttacagacctttg ttctcagtag 46800 catgtagctg agctatttac catcccagcc cctatcctattgtaaagaga aaaacagaag 46860 gaaaattttg tgtcataggg cttctgtttc tactgatattagatgtataa attcagatta 46920 gtcttttctg tgttacacaa attaaagaag tgaatggagcaggaatctag taggagttaa 46980 ctaatgttta ttagcatcca aatactgtgc taattacaatgttttatctg tgtaatcttt 47040 gcaataactg tattagagat agagtttaaa tatccttttttcagatgatg atgcgtattc 47100 aaaagggtta aataatttgc ccaagacccg tagtggttgggttagggttt gaatctaggc 47160 ttgttgaact ccaaatcttt gctcaaatct gtagtcttttttctatataa aattgatttt 47220 gtaaattatc ttgttttatt ttaagaactt ggggtgttttttaatagact gaaaatttca 47280 ttatcctgag atttggtgat ttactccatg tgttttccctttttaagaac ctagctggtt 47340 attttgaggt tagaagttaa cattatgtgt atgctttagattcagtctga tgctttttca 47400 ctatacctaa tagcctattg caactttgtt cagaattccttttacaattt ccacctgtag 47460 acttgaagca ttaactattt ggtctggctt ttgacaaaatgaaaaaggag gggttggagt 47520 agtgaggtga gaaaatgaag gaaatgaagt tgccagctatttgaaaaaga atggataaga 47580 ctatatggct gaaaaaagat aggcattaat tttaaataaatggagatttg gaaatttgcc 47640 atgcttttaa tgagaagaac ctgatagtta cggctttgttttgtaactac atatacataa 47700 aaggcttggt gttttgtagc aagttctaca cttcaagatgagcataaatt ccagctctat 47760 tcatggtttg atttagggta aatttgtcag cctccttttctcagaagcct tcatttgcta 47820 ttatctcttt cttgggatat tgaaacaatc aaaaatgcaataaaaagttt attttttcat 47880 ttgttctgcc aacagctatt atgctaaaca ttctaaattcatcctattct atatggtggc 47940 ccctggtttc atcttgctat tgagcatctg aaatatggctaatgtgaatt gagatgtgtt 48000 gtaattgtca aataactgat tttgaagact tagtatggccgggcacagtg gctcatgcct 48060 ctaatcccag cactttggga aactgagatg aatggatctcttgggcccag gagtttgaga 48120 acagcctgcg taacatggca aaaccctgtc tctacaaaaaatacaaaatt agctgggtgt 48180 ggtggcgtgc acctgtagtc ctggtcactt gggaggctaatcacgccact gcacattcca 48240 ccctagggga cagagtgaga ccaagaacct gtctcaaaaaaaaaaaaaga acttcatatg 48300 gaaaaaaaga tatcttttta ataatgttta aatattgattagatgctgaa ataatacatt 48360 ttggtacatt gaattaaata taacataata ttaaaattaatttcccctat tcctttttac 48420 cattaaatat tttagtagcc actggaaaat ttgaaattatatctgttact ctcattatat 48480 ttcagttgga cagcattact tcagatgcaa agatggtctgtaggtattac cattgctgct 48540 gctttgtaga agcatctgtt ctagccctga agtagaggaatagatggttt ccccatggcc 48600 tgtgggcaga actgtgttat ataccccact acaggcgtttttacctactg tggctgtcta 48660 attagtgttt gtttgtgcct tcaaaaacta ccactcttgtggactagctt aagctcagat 48720 ggaaaaaaga aattggtctt agggagtggg tgtggggagatgatttactt ctttataggg 48780 aagtgtctta tagggataag cctgaatact ggatattcctttaggaaata gaagtagacc 48840 tgatggttgg atgaagatag agcacaaacg tctttattatactgtatttg gcctcaggcc 48900 atcttatcag agaagttctg ctctggcaga tatccatttaaggaaaaaag ggttttaaag 48960 ggcccaaaat ttattagagc agtgtagatt aatgttttgtctacatagac tgttatttga 49020 attttctttt taaaggttca tttccttcag aattaaagttgaccctaagc tcaactcaca 49080 aattcctctc tgtggctgct tttgactttg ggctcacttctggggcagat gtgatttatt 49140 ttcttgtgct tgttattaat ccagaaatat aatttcagcatgggcttgga atagtgctct 49200 gggacatgaa gcagaagtga acaatggaag ttaggtggatatgagtcaaa gaaaactgac 49260 cacactttct ctcagctctc ctttacagca tgatgagcctcaagagctgg gggaggtttt 49320 cattgctgtc agtgctccca attcagtaaa aatgtttgatgtgagttggc aagtacattt 49380 gccagaggtg gtgcatgagc tccttgtgtc tgggaatggagattttcatg atggtgcttt 49440 tattttgagc tagatcttcc tgtctcagtc tagcctctagctggtgatga attattctct 49500 agagtaaggc ttttctcaca accatttttc tacttttttttttttaggac tatagggatg 49560 gaggtttttt gattgagaga gaatactact taattatatatggtatttca aagctgctct 49620 cttttatatt ctacatggag gaatgggaaa atatgtctcacatttcaatt tttctgctgt 49680 aagcctgatt gtgagaagta taataagcct gttttctgaattctacctcc tcaccccaaa 49740 ttcggtttcc tatcagttgg gcagactgcc acagtgctaacctgaagcac ccttgcccca 49800 gatgtcccag agttgattag tggcgctgtt ggtggaaacaaggacgagtg gatagtctag 49860 ccccacatgc aggaggtgag actgcaggca ggatctttcctgccctgcag tccatgactc 49920 agggtgtagc cttggctatg ttacaggcac gatccagtttaacttcatgg ctctctaagc 49980 ccccatggaa cggttgttag taggttcaat aatttatctattggacctac ttacagatat 50040 ttattgaaca actactacat ggaatatatt atgctagatactgtaagaga cacaaagaaa 50100 aaattcctat tttaagagtg gtaaaattga ctaggaagacataagaaagt ccacacataa 50160 ccacactagg aagcagaagg tgtgtgtgaa tgtgaatttgaaagaaacaa tggtgggaat 50220 ggggaagcat ctaagtactt ggtggggtaa ggaaggtgatcgagaagagt gaacttctgt 50280 tttgagtctg agtaatggat gggaactgtt gacagagaaggagaaatagg gtagaaatga 50340 tggtaagttg gagatgagtt ttccaagtga aaaacatgcagcagtaagac ccaggagctc 50400 agaatacagg cagggacgag agactcaggg ttaccatggaatgtgtcaag tctgtgaggg 50460 aaattggatg gagtgataag aacagaggat ttggacccgaagtatgagaa acatccacac 50520 ttcagaaaat tgacgaggaa ggggatccaa agaaggaaagtgagaaaagg ggaggtttga 50580 gaggtgggaa gagagctcgg ccatgatagc atcctcaagttaaagggtac agccttagaa 50640 gcttgccctg aactcaactc cacatgcctt ggtggcagctcctgtaatat tttacttctc 50700 ttactgcgtt atttgttcag cttgcctaag agactatcagcctctggaag gcaggaaact 50760 gctcttactt attttgacat tcccagaact gattttaacaaaccatggtt attaataact 50820 gacaaccagg agactcaaat ataaatgata actgcaaaatattcattaga atatttgagt 50880 cattagaaat tttctgaaat acagttttaa tacagacaagactaagaagc tagattgagg 50940 aagttaaagg aaaaagtagg ctttggggag aaggaaggggcaaaatgggt ttagatcact 51000 atgtaagatt ttgcacaact gaaaaggaag agcgagggagacacaacgca gggttatggg 51060 agacagaaac tttattcatc tcattgacca tagagcttggctacactaag agtaaggcag 51120 ctcaaagggc gttggctaat gaagtggatt attgggaggggtttactctt acagttttta 51180 gtggggaata ctttaggtaa gatgtctgct gggaatgggaaaggaagaag atgggaaagt 51240 tatagtcaga gagtggaagt accaagtttg agatcttggactagaacaat gagtgtcttg 51300 taacagaggt gtagaatgtg agcgtgcttg tgatatggaatggatgcgga ggtctgtaag 51360 gttgagaaaa gtaagaactc ttgactcaga tgaccttactggtcctcaac ccagatgttg 51420 atatcatgcg agacaggaga ggaaagagcg agaacataagccacatgctt gtcattctag 51480 gaagaggaaa ttggtccacg agaacgacag accatggtgtgataatttat attatactat 51540 gtcttttttt taatttttaa aggacttcaa ttcatctatctacttttaat gtcagaaatt 51600 agaatgtatt tattgatttc tttctctcga agatgaggaattagcatact tctcatagct 51660 ttcactcact cactagactt tgtataaata atctgggtttcaatatttat attattgtta 51720 tcaaatttat aaacatatac atattcttta aaattattcttgatatttac aattttataa 51780 cccaatttat aatattttca acttagttgt atgtgctacttgttttgtgt taactaccag 51840 tgcaattatg tcacaacatt ttcatttctg agttccttattttcattaac attttagctg 51900 aataagtatc tttgaataat tttatttcag aggagttacatgttggaccc atttggttag 51960 tcaaaaaatg tcttttgatt tagttgcctt catacacaagttgtatcttg actagatatc 52020 aaattcttgg gtcacgaatt aatctcatca aattcttagacattgttcct ttgtgtcttc 52080 tggttactat tctagggaaa tgcaatgcca gttgagtgtttcttcttttt cataagtaac 52140 actgggctcc ctgccttctt tttcatttgt tgtagagttgtcaggaaaac ctattatctt 52200 tgcaattagg tcttcattct ttcttctgca aagctaaaatctctcttgat ttttaatctg 52260 ttcatttttt gttttgtgtt ctgtgagagc tctcttaaggttcttcttta taacactgac 52320 ttatatttct tcattgtcag gtcagtcctt tatagcttcttctgcctgcc tctcagcccc 52380 tttgatcaga gactaatgtc ctatattatt ttttatttctttgtagaacg taaacatgtc 52440 taaatgtctt gagtgtacag gagcagttct ttctaaaatgtattagctcc tgtaagtgaa 52500 taattttccc agtgtgcttt tcatcaatat cttaagcattacattctcca tgttttatgt 52560 tgtagaatct tttaataggt gccatgttgt ttttttccattttactcatt ctgagcagag 52620 taactgcctg cccatagaca ggggaggtgg gtgttcccctggatcccctc actctcaacc 52680 tggctgcagc tagtttttcc tcctggtgca gtgaggggcttttatgtaaa ctggctcaac 52740 aacccagcag cctaaggtgg ggagagccag agagtggggcctcctagcag gagaccaaag 52800 cttggcacca ctttcttctc tgtggcgggg gtcagctttctgaatgccca gtactggccc 52860 agcctccatg gcaatccacc cagtggctga gcctggatgcttttcagtgc tgctgcctcc 52920 ggaatctgcc ccagatgcac tgcagtattt ctcattgctatagtggccaa tagtggccac 52980 aggagcctgg actctgcagt tcttgaagtt tttgttgacctggaggggtg aggtggagtt 53040 cagtccattc tcctcactgc agcagagtgg actttctgaaacatgcattg gatcgagtca 53100 tttctttgct ggaaacactt caatgggtcc tccttgtctttggatttagt cccaaggctg 53160 tgcatgtaac tccctctcca gccacacctc tcttcactcccctaatgcat ttcttataat 53220 tcaaccacaa gaattttttt tcattgccgc aaatgaaccatgctgtctct tcaactcaag 53280 cctatgcttt ctgcctgaaa tactccgcac tccaccctttctctacacac tccctcacac 53340 acttctcccc tctttcacaa cctggcacgc tcctcccaggccttctctta aaagttacat 53400 tctctagttc ctctctattc tccctactct gtactcccaaggcaataggt atctatgaaa 53460 gcacctgtca ctctgtgtca tcattgtttg tttggttataagtctgctgt ttgtaagttc 53520 tgcaggcaga agccatggct gttttgttca ccattataatcccattgtgt atagtcagag 53580 cctggcacat tgtgtttgtg tgcgtgtgtg tgtatgtgtacatatatata ttttagataa 53640 acgaatgaat gaattacagc attaaatttt ccagttgttcccagcccaac ctcatctcca 53700 aattcacagc tcttcttacc tttctaagat actttagtatttttcttcag tgtgtaccga 53760 gtttgtacta aatctgtaaa attagactaa ttatatatgaagactatagt tgacaatcaa 53820 tgtaaagcac atatcatctg gaacctagaa ataactcagtgcatatgttt ttactgctga 53880 ggttagtagt gacgttgggg aataaaaaga aaacccgctctttctcccct agtttcctgt 53940 cagtggcagt gtttagaaga ggaggactgc aggagctctaccgcagaggg ctgactgggc 54000 tggtgtgttt agggaaaact caggtttcag tcaaagaaggaagttgtaca ccttaatgta 54060 gagttgccct tgaggcgtag ttgaaggatt agtgggcaaaggcaaggtga atctgggtgt 54120 gatacaaatg agatagagtt cagtgacaca ggaaggggactgggatgagg ggtttgcgtg 54180 tgaactggtg gtgtagacaa gggtgacgga cactttgggtaggaacagaa tagagatact 54240 gttgctatga gaagggtgag ttgaaagata attattattttgggattagg ataacttaaa 54300 taagagaacc ttaagcagat aaacattcat ttaacgaatatttatttaag cttgggattg 54360 tgtttcatgc tggggattca gtgatgaggt aaacagctaccccccatttc ttcatggcac 54420 tgacagtcta gtgagaaaca caggcataga caatataattctccataaaa ttttgaactg 54480 tactaaatgc cttgcaggaa acatagaatg tatctggagagcatattaat aggcagaact 54540 aattgtgttt agtgacaagc aagtaaggtc tctttgagaaagtaacattt tccatataga 54600 caatgagtag gagttgggga gagtgaggat aagactatcccaggcacaag gaataacgtg 54660 tgctagccac tgaggaggga aagaatgtga tcattttgagaaatgaaaaa agcccagagt 54720 ggctggagct agtgatattt catggttaga gcctaaagagataggcagaa aacactggta 54780 ggtgttagaa ttttatccta aatgcaaaat aattcatcaaaggatttgaa caagcgagtg 54840 gcgcattcta ctttacatgt taagatcatc ctggcggctctttagaaaat acattggtca 54900 tgatgcaggg aaaccaggcc tttgagaagt cctgaaaaagatgatgctgt ctagaattag 54960 tggaggaatt tcttgcattt attctgtgta tgaatatcctttccacaact acattatccc 55020 cttagccaca tctatactcc cagaccatcc tctcagtaggcaggccctgt agacagcagt 55080 gtttatacag aatgtttcac ccaactttat tccgagcaggtgatgggaca ctcaagcttg 55140 ttctttgttc ttcagctcca acttcagcag cgtgatagaaagtctcctgt gtatggagct 55200 gaggttttgt aggatagaag tcccaggaat gatctgctctgacacagaaa gagatgaaac 55260 tccagaactc aggggcttgg gtgactgctg gttggaaaggtatataggag atggagctct 55320 cttgactcag ccatagagga gaaggtagca gactcagtgcaagggccagc caagctcatt 55380 ttttatttgt tcctcttgtg gagattgttg ttgcaatgagagttgaccct taactggacc 55440 agtaatgtga tcagactttt tcccacagac ttcacagttgagagatgact ggaaaggatg 55500 agagtcgagg ctcagcacct tacttttttc aattcctttctcttcttttc tttctttctc 55560 ttgttgtccc cctctatttt ttacccatca cttgtagctccaaatcagat tagtggtttg 55620 tgggtggggc ccttgccttg agagagccat aatgagctgtttgtgtccct gcgtgttaca 55680 gctcataaat gctgactgtt tcatggtctt tggctcacttggctgaagcg tggtgctaat 55740 gaggcccagg ccatggtttc tatttcagca cagacctcttaggttcaccc tgttatattt 55800 actgccacag gctgtgtcca taattttttt ttttttttttttttgagatg gagtcttgct 55860 ctgtcgccag gctggagtgc agtggcacga tctcagatcactgccacctc cgcctcctgg 55920 attcaagcga ttctcctgcc tcagcctccc aagtagctgggactacaggc atgcaccatc 55980 atgcccagtt aatttttgta tttttagtaa agacggggtttcaccatgtt ggccaggatg 56040 gtctcaatcc cttgacctcg tgatccacct gcctcagccttccaaagtgc tggaattata 56100 ggcgtgagcc accacgcctg gctgactgtg cccataatcttacttgttca gcctgaagcc 56160 agaggatgga gcaacaagaa aagatcagca tggaggtgaccctactcctg caaaaccaaa 56220 acagtatact tcctttgcta atggtggctt ggtaatgccatgtcatcttc acttgtagca 56280 ctctgagttt cttcctcttt ccctacatcc ctcaacagtcagctcagatt ggccttcttt 56340 attttgaaaa ctaacttgat ctgcagcaat ctcacaccccaaagtcttta tcagcactga 56400 gctattttca cttctaatta ttttgccaac taactgtgcaaagttgggca agttgctata 56460 cctctcaggt cctagtttcc tcatctgtaa aatgagcatgttagattaga tgctgtccaa 56520 ggtcagttcc accactgaca ccaagtgtct agtacagtggctggcataga tactgaggtg 56580 aatggcagac acagtccctg ccctcctgga gcttatagcgaaatggggaa gagactgtaa 56640 agagtaaata aatatgaaat cacagctcgt gatacattccaggatggatt gaatggaatg 56700 atgcaatggt ggagaatcat gaggagatgt gcatttggagacctactcta ttgataagac 56760 aggtacctcg ccatgtgaat aagcagggaa aaaatgtttcaggtggacag agcagcttat 56820 gcagcagcac tgaggcagga gagaacttaa tgtttgaggaatcaacaaaa gttattgtgg 56880 ttagtataaa ctgtacaaag gagaggtgta gatttgagtatatttacgag gcaaaagtga 56940 tcaaccagaa aattgtcagt agttacaggt ggcactcagtgtttaaggta tcagccaagg 57000 atatgctagt ttgacatttc atttcagatt attccagagtcattgtggca ttagagaggg 57060 tagtttttaa aaacaagtta tcttctttct cttttgcccataggaaataa gagcctaact 57120 agtccactgc ctcagtcctg atgttaaaaa aatgacagtagcctgcaata tctggcaggt 57180 ggaaattact gagagggata attattgctc ttgaaaacttgcctaactga gctttgtgca 57240 acattttggg agttcaccag caaagtttgt gtgttgacacaggtgatgat acatgctgac 57300 acttgtttgt attgaagatt tagtagcttg gtaggacatggaaacaatac agctctcctg 57360 tttcaccaaa ggcttttatt tgctaggtag cctataacttagtgctctct cctgctttcc 57420 ctactaacat aaagtcattt gcttttaatc ccttctatgttctattggcc aacttacctt 57480 caaacacagt atcgatcatg tctttcctca tcctcctttacttgttgtat caagttccga 57540 attcatactc gaagtcaaag cctcaatgac ctcccactttttcttcttct aaaaccccag 57600 ccaaagaata cactgcagtt attttaatat gtttaagacttaatcctgta ttcctctgta 57660 aatcttcaac caccagccca ggctggtgct ttcttgcaccagtgggataa gctcaatgac 57720 cgtagagcat acacgttaag acctctaggt gatcgccaagaacttttccc actcaaatat 57780 ctagtgacct tctgccttta ttcctttcta atacgagtgttccttaactg tcttatgcac 57840 ctatgccttc tcagggtgct gtaagaccct gggatcagcgacctcatctg tagattgtgt 57900 tgatcacatc cagggctgaa tactcaactc tgctgcagtcaaagagcatc agatttgcag 57960 tcagatcaat ccgggtttca atgcagcact ctcactcaccaaccgtgtga actttggaag 58020 attttatttc ctttttttta ccttcctgaa cctgaagttttttattttta gtctttagca 58080 ttggttttct cctgtgccag gagggtggtg acgtgaagggagttgttata tcatagcaga 58140 tgttccactg ggaattttag aataagcaag ttttatggccaccctggtta ctgtttttcc 58200 tattctgaat tttagaacca gttgatagaa ttttaatagggagaaaataa tctggcagaa 58260 ataggactag gaggaaatgt gaatttgtag aaatgtgcctgaaccttaga gaagtgtgat 58320 ccatccagta gctaattact tataagccac tatcttcttggatctctaaa tttcctgtcc 58380 ctaacaagca ctatcgaaca gatgcatggt ccaaaggtgttcagtgaagg cagctaatct 58440 tcaaaattca gtctcattca aaaatcagtg gactgtacctaaaaatacat ttccctttta 58500 gacaggagtt tgcagtcaca agtgtttcca gtaaccaattgtttgagtca tgatttgagt 58560 cctcacctcc tccatcctgt ccacttcccc atgtctgtgtgcccttggga agcccgagac 58620 atgcagagat ggcttgatat gttcaggttc tcacctcaggtctttgttaa gaattttaga 58680 gtgaggcagc tccagttcaa atctcaactg tccttcactcactggcttac aaaggcaaat 58740 tattttacct ctttgaggct gtttcctcag atataaatgagacaattttt atgggagccc 58800 aagcacagtg cttgcttctc agtaggcagt caatggctactggtttcctc tcccaagagt 58860 gccacatttc ctttccttta tttacctagt ttgtcttgctacattatttc agtgtctgtt 58920 tattatggtg cataagaaat gtatacaaaa tagctttaaacacattgtgt cttatagtac 58980 ccaaaattat cacaaagcct caataaagaa aaatgacatctaaattgttc acattcagca 59040 agtcagtttg gttggtgggc agtggatatt tgagttagcataagtataat agatgagact 59100 cagcaatttt catgttgcag tagctcttca cctacttcttggtcatgttt tcatttctca 59160 atcccacctg catcctaata acacagctgt cattactttgaggtaccatt tctgctagca 59220 ctataagaca gcaaagtttc tgtaataaat gacatcatctaccctgaggg agattattgc 59280 cggtgagctc actccctccc tccttcactc tctgattccttcactcactc actgactccc 59340 tccctccctc cctttcttac cttcctccct ccctttctcacctccctcca tcttttactc 59400 tctcacgcat tccctccctc gctcactcac tccttgcaatcactccctca ctcactacct 59460 cagtgttctc actttcttat tcatcttctc ccttccttcctccctcattt ccctcattac 59520 cctcattacc tcccccattc actccctcat ccctcactcactctttctgt attaatactt 59580 tgggatgcag ttgacacagc ctcgtttcaa attagaaagacctgttgcaa gaggcatctc 59640 tcagaatggc tatgccactg agtaggctta ctagtagttgggcttcagta gcaattggaa 59700 ccaacaactt gaatgccacc atgttttttt ctctttgtgcacatcttttc tatttttctt 59760 tttttgagac agggtcttgc tctattgtcc agactggggtgcagtggcat gatcatggca 59820 caagtgattc tcctacttca gcctcccaag tagtgcctgcaggctggtct ctaacttgtg 59880 ggctccagtg atcttcctgt ttggcctccc aaaatgttgggtttacaggt gtgagccact 59940 gtgcctggct ctctgtgcac ttctgattca cccatatcactttctctgca gaccagcctc 60000 ctcatctttt tggtctatgt ggcaaagtgg tcctactccaataatttggg agagatacag 60060 tgatgtagct tggatcagat gcccatcttg ggtcagtcagccatggccag gagggtttga 60120 tcatgtaaag acatggttgc tccttcaaga attatatggctggagttagg agaagagcat 60180 ggaagggtta tgccgagcac ggagttccgg agacatccacatacccatcc tgccatccta 60240 ggcagttggc tcttgctggg aatacaaaag aacccctctaaactgcagcc aagtagtggg 60300 agacagatgt gcaaccaaat aagtgtgata gagtgatattggttctatag taatggaggt 60360 acatgttaga taggaataaa gaacaggcag tcagttaggttaagtcagac acaactagca 60420 tccgcgagta atgatggcta acaggccttc tacagatacatgagccagtc aaattgctag 60480 gtattctggg tctagagata gccttggcat tttccaagcacaaatattgt gttctttttt 60540 tattaggagg cctctttatc agagtgcctt ttaaaattatttgttgagtt ttatttacaa 60600 aggaagaatg aaggacacct tgacatattt ttattttcaacaacttgtca tgttttagag 60660 aattcggatg atctaaattt tagttccttt gatagctctctaaactgttt ttaaaagacc 60720 tcaaacaaaa ccaatcacca ctttatttta tagctcatatgatctggccc tatactgttt 60780 ttgttaatca cagattcctg gttgtaagtt gaagacattatcttgttctt tgttaaggga 60840 tgtaggagcc tttgccttag tctccattgt tgctatagtggtgacctgca acctagccag 60900 atgtgggctc atgtttagct tcctttagtg tgatctttttcaggcagtgc ataccagtcc 60960 tttttccatc tcctagtaca ttgggtggac tttctttgtcttcttccaca aatccccatc 61020 ctttaagtat gggtgcctca ggctgagcac agcttgctccagtctcatta ttcttggcct 61080 tgcccttcac atccatactt caccagagat tttcaacatctttcatgtct tctctgtacc 61140 ctatgttgct tgccctaaca ttttcaaaaa ttagactttttattttcaga tacttgtagt 61200 tttgtgcagt tataagaatt aatatagaaa gatttcctgtaccctttatc aagggtctcc 61260 cagtggtaac atcatcttat ggaactgttg cataatattacaaccagtgt attgacgttg 61320 atacagtcaa gatatagaac agttacccta atgtttggcatataggttcc ttctcattat 61380 tttttcttac tcatttttat actcttctct ccttgctcttcttacttata tgttcattga 61440 cccagggatt agttaactcg agggtcctac aggttttaccttctctttga taagttggct 61500 aaaccctaat gttgttagag tttctaacgg tagtaactattcatcttgaa acgatttgcc 61560 tggaaaaaat atcatttaga tggctcaaat tggccttaaaattcatgtga agacccagcc 61620 ttctggagct ctgcttgcct taatttcaga caggtcacatgaagatgaag agtgtggggg 61680 gctggtatgg tactagtcct ttgagactca agagccctaatcaaaagtaa tttcccccta 61740 aaaggacagt tctgcacaga ggatgtggat gaatgcctgctgcagcccaa tgcctgtcaa 61800 aatgggggca cctgtgccaa ccgcaatgga ggctatggctgtgtatgtgt caacggctgg 61860 agtggagatg actgcagtga gaacattgat gattgtgccttcgcctcctg tactccaggc 61920 tccacctgca tcgaccgtgt ggcctccttc tcttgcatgtgcccagaggg gaaggcaggt 61980 aggtagcaga aggacggatc tagaaacagg gaaggggcagggggcattca ggactctgcc 62040 ttctctttca tcccaaacag catattcttt gagttaccatctatgggaac atatcactca 62100 ggactgttcc agaaatgtaa cctgctagcc aaagtgctatgaagataatc ttcctcttta 62160 ggactttagg ctctcttctc aactctacat actttagtttttcattccaa aaaaggaaag 62220 ttagtaacta ttgtctttgg aacatctgct gaagtccttaagatcttaga ctgaaaagca 62280 cctcagctat taagataaaa tggctaataa atggatccacaggcttcatt ttaagaagaa 62340 accatctatt tactcagaag aagttggcag gagagaaacatttaagtgcc ttctaaccct 62400 caatgtctat gaaaatattc ccctcaggga agatttttacatgctacaaa gttaacattg 62460 ctctagcctc tcaatttaac aactactagt tctaggagaaggagagcaat gtaaggagga 62520 aggtgatggg agaagataat ttagctggga gaactttctgcccagcagtt ttgaaatgtt 62580 ctttcatgga ggcctttcca aaagctctag ctccagctttgtggaagaag ctttagttac 62640 ctcttcttaa gttaattgca gactggaaaa tccagttcccatgtgaacta tataaaacag 62700 gaagcctagt aatatcttca gagacctggg gccacctgaatcaggaatgt tgatgctatt 62760 aaaagattac tctagtagag gctgatggtg ttcaggtggccccatgatca tcaggtagca 62820 taatattggg gaagtaggat tccggataaa atataggatgtatggaatat ttgagacata 62880 ctaaaaatta cttcttgttt atctaaaatt caactggacatcctctattt tatttgctaa 62940 atctggcaca ctatctggaa gatagctcca ctttcctgtctccgtcttct tatcttctgg 63000 cctgtagatg tctctgtcct ggaggagagg tctagctgagagctcagggt cagagtctag 63060 ggttgcatct aggtgctttg ctggtggcca agcatgtccccaaggtaggg tcaggcaaag 63120 cagaagttgc gtataaatct ccatgttgga ttctgaggaactgatcatga ggttttccct 63180 cctgggtgaa agagaaagct cttccccaag tgagagacattttctcttag ttgtcctttg 63240 ccccaccctg tgacaggtct cctgtgtcat ctggatgatgcatgcatcag caatccttgc 63300 cacaaggggg cactgtgtga caccaacccc ctaaatgggcaatatatttg cacctgccca 63360 caaggctaca aaggggctga ctgcacagaa gatgtggatgaatgtgccat gggtgagtaa 63420 gcagaacttt ttctgttctg tgaagtgtct gttgagctcagcaaatgttt aaactgaagc 63480 acattttact gtagaagcaa attacataca cccaaacctcagattactct tgctacatgg 63540 ccagtgttta caagtgacct ttcagcatag ccataaccatatattccatt ctggggatta 63600 actgtatgaa acgtatgtac ttgattctta atataaagccctacctcaat gacagtttgg 63660 gcagaaactg caatagcaga aatgcataac agttgaggatactccgcttt tatttctttt 63720 taaattaagg aaaaaaaaat agaaaattgg gaatgatattattgaaggcc tggccctgat 63780 gctataacca tcagatgctc ttctattgtt cacatttgtggaaactcaac tgctttgatc 63840 ccagtaattg gtacttgtgt ctttcagcca atagcaatccttgtgagcat gcaggaaaat 63900 gtgtgaacac ggatggcgcc ttccactgtg agtgtctgaagggttatgca ggacctcgtt 63960 gtgagatgga catcaatgag tgccattcag acccctgccagaatgatgct acctgtctgg 64020 ataagattgg aggcttcaca tgtctgtgca tgccaggtaaatgggcccat cagcatgttg 64080 ggtctagaga ggagggactg ttctgaactt ctcttggtagagcattcagt atactattgc 64140 agtctgatga gctctgtcag catttgattt cctttgctgactggggacta gaggaagtca 64200 gggttcacaa gcaaaccttc cttactcaaa gagtaaggcagtgctagaat tcatcaagct 64260 atgtagacca aattaaacaa gcctgttata tcaaaatccacatgtaatag aaaatttcaa 64320 aaatgattta ctgtaagaat cctttaaata gcaagttttcctgatacctt catgtaatta 64380 caaataccat accattccat ttatataaat aaattttatccacaattata taggaagttt 64440 ccatttatct aaagtgcagt acatgtttag ttgtattctggagctcttct gcatagacca 64500 tgtctttaat tttgagagga tacagccaaa tccagcttgaaatgatggct aacattggct 64560 gctctgcctg attaaggctg ccggccactc cagccttgaaaggcactgct gaccttgaac 64620 tgcaccctga aggcaacaac agctccagct cagcttgtctttaggaacat agagtgggga 64680 cattttgggc cttccacagt ccagaaagta tggatttatcacagaaaatg tggagcagtc 64740 cttgtttata tgctaggtct tgccaatgtc ttttaatgtaatcccttccc gcttttcaca 64800 gtgacatggg tatttcacag tatgccctgt cctgcttgacacttggagtg actatttctc 64860 atcatggcag agttcctttt ttctctgcag aaatgtgtgtactcgcaaag gatgtagtta 64920 tcattgcaaa tttgtgcttc cttgtctgga atacacctttctctctcttt aagccacttg 64980 tgtggtctgt tttgtttagg tttcaaaggt gtgcattgtgaattagaaat aaatgaatgt 65040 cagagcaacc cttgtgtgaa caatgggcag tgtgtggataaagtcaatcg tttccagtgc 65100 ctgtgtcctc ctggtaagtg cccacgacct ggccctgttttctcttaaag cacaaagcca 65160 actgaccaca gggaagagag cagggagaga atgtgtgtgggctgtgcatg aggaaatcat 65220 tagagtaagt gcttggtgat aggagtgtgt gtgtacagaaatagagaatt tgttttagct 65280 actatatctt acaagtcctt tgtggccaaa agtccaagtctattcttgaa tggcccatga 65340 cctcttcgtc tgtgtatata tttcagaatg ctcatacctcacagaagcct tagttgtagg 65400 ctattcggga ttttattttt ttcccatggg tcatattcttgaaggccctc tgtgagactg 65460 ttatttttag acatttcacc catgagttca gaaacagacaacacatgcca atttttccaa 65520 gattctctgt ctctcctttt ttttcctcaa gctatttttgaagatatgcc aaattgtcct 65580 ccatagattg catcttaccc cattgtatct tcagagagaaggtttctgtt gcctctgaag 65640 ctacagttac ttgtttgagg ttccacctat aaatttcacttcctacatcc ttcatttaga 65700 agtgaagtct gtctgtgaag aagcaatggt agagacacaccacaagtgcc tctgattccc 65760 tgagtgtcac ctggttattc cagttacctg ttggggagaataataaagca taggcttatt 65820 gacccattgg tataagccct gctagtaacc taacatcattctcatggtaa agggagacac 65880 aagataatcc tttgactcat gttaccactg tccctttttgaggtttcact gggccagttt 65940 gccagattga tattgatgac tgttccagta ctccgtgtctgaatggggca aagtgtatcg 66000 atcacccgaa tggctatgaa tgccagtgtg ccacaggtaagattcttctc acttttcatc 66060 tccttggtag gtccatctga acaaaatagg gtgattgtccctgtcctctg ctagctgctg 66120 tgccaggcca gtccactccc agaggctcct ctgtgtttaggaagagctgt ttctaacaaa 66180 ttttttgctg ctctcttttt ctgcagcacc tgcctcctccaccacaacag tggtctggcc 66240 cagatatttc catttggtat ccatgtcttt ctgggacagattttggcagc agtaagtttc 66300 cagctggcta gattgactca tttaggaaag agaagaaatccagttgaaag ctatttttca 66360 ctggggcttc tcagcaggag gcctggctct gtgtttcttgaagcaagtga ctgaaagaag 66420 gcccagaaat ttaaaggctg aatgtgtaca agtgcaggggtattctgcat aaaagacacc 66480 aaaataggat tgagagtcaa gtgtgtaaat gacaggagagagagctctca ctgggaaagt 66540 ttgggtcctc tttgctgact tctgcaacta gagtcaaaaacgatctgtgc acaaatattc 66600 tgcatttctg ggtttgtaat cacattttgt acttgggattggatttcagt aagccagatg 66660 ttccctatta cacatttcct tttacacagg caaatattatacagaaaggc tagaaattat 66720 tagaagtgaa cttctttatt atctgactgc ttgagaaagaagttatttat gtaattcaag 66780 acattaattt tagagaatgg agtcttaaag attaaaggcatcatgcatat aatttatttc 66840 tgatcacaga aaagcaaatt taattgtgaa aattaatacaaaaagttaat attttaatta 66900 ttagacctac ttagtttagg acaactcaga tgagccactttctttctttt tgcaaatagt 66960 taatgcttgt cagagaatcc agattttgtc ttcccctggccagattcttc ttgtagagga 67020 taagtttcaa agagattaaa aacctttagc tcaggtttaaaagtgggggg aggactcctg 67080 gagtctttta gggaaagata taatgtttgt ctatttacacctcgaagaaa ggaaatctta 67140 cttgtctctt tttcttttaa attctcataa caggttcattcattttattc aaccagtatt 67200 tatttaaaac tactatatat gcatactact ctcagaggagttgagagaag taaaataaag 67260 ctctcttacc tccaaagtca gaattgggga cataagatacatgaaatcat taacctgtat 67320 attggacttg ggaagaaaga aatacaaaga taaatatataaaggaaaaat attttgagct 67380 gttatacctg aagcctagta agaaatgaag gtgcagttaaagcatctctg caatgaaatt 67440 attgatcagt tgcgaaggta tttcaattct aaaatttgatttggaaatct ttattttaag 67500 cacctctctc caggcatact ttctaaatca atgcagatcgtcgttttcta ctgctcaaga 67560 cctgcagtat taattgacca cacatcgaat gtgtagagtaaatggatgta gctacacttg 67620 tgttctggtg tggaatatct gccttggtct ttgagaatccttgataaaat tctttccaaa 67680 ggtttcactg gtgtgttgtg tgaggagaac attgacaactgtgaccccga tccttgccac 67740 catggtcagt gtcaggatgg tattgattcc tacacctgcatctgcaatcc cgggtacatg 67800 ggcgccatct gcagtgacca gattgatgaa tgttacagcagcccttgcct gaacgatggt 67860 cgctgcattg acctggtcaa tggctaccag tgcaactgccagccaggcac gtcaggtaag 67920 cccactctgt ttatgtttgg tatggggttt tcactgtttctcaaatctgt ttacacagat 67980 gtttcagact gttggcatat gctcagatac ctaaaaaacagtcctgattc tgagaagcac 68040 agccaaagcc acaaacataa atgtctcaag gcaaatggccctagccagtg gagaagtcag 68100 agacaatagc tctgccaatc tgatgatttt gtgtgtgtgtttgtgtgtgt tttgttaaat 68160 agaggcatac cacttcaaaa attaaaatta atgatttaaaattgaaaacc ctcttttttg 68220 tggctatatt atcttttttt ctttaagttc caccccacccagattaataa attcagtcct 68280 tccttccttc cttccttcct tccttcctac ctaccttccttcttcccttc ttcccttcct 68340 ttaatgtatt ttctctttca cagttcagtc tctttattgtgacatgtttc ttcagtcact 68400 tgtctctggt ggggccttag ccatcttggg ggattctaaggaagaggaga taccttacaa 68460 cacaccacag taactaaagt agaacgcaga gctgtcatttcctatcctcg gagttcatca 68520 ttgttctctg accttggttg aatgactcct tgttggtcgtgaggacatgc tgctcctttc 68580 tggggggatg tggaattatt ttctcaggca gcagcatctcacaggtttgg atggttctgt 68640 tttacttatt agatgaatga ttatggtcac aactatggttagtggacaga gatgtactat 68700 gttccagatc aaaggaaaat gtattagtgg aagattaaccaatgtgtgcc tttgttagaa 68760 ggaaacctgc caatataaag gagtgattta ttgagaactcattctggatg taaggaaaat 68820 gacctatgta gagagcagag gtggagaggg tagttagctaccctcatttt cttttactgt 68880 ttccaaatcc atttcttcaa atttgtcaaa ggctctggtctttacccaag gcttcataca 68940 tgccaagttt gaaaacaaag tcctttttga attgctcaagcatacgaaaa acatcccatg 69000 ccgtcacagc tgaaaatgcc tttccctccc cacatttagataactcacta aaagggctga 69060 atagatcttt gtgaaaactt ccaaaagcaa tttgcttccgggctgttcct cagacatgag 69120 aaattatagt aggaaaagaa atttgagaga tttacaagcaattgactgga tgtctttaga 69180 atcgaggggc tttagacttt gctatcattc ccatcataataaacatactg gaaacctggg 69240 tgagtggtgg gttggcaagc actttgtttt ctctggcattatatcattat accaggatat 69300 atcaggtgtt ttctggggag gacagagagg gtatggtataagtagctgtc actgagaagg 69360 tgagaagtca atatgagtga tctgtaggca gcatggaaaggaaggcccgg aatctgtcag 69420 aacctgccaa agatgttcct gcagttattt ggagaattaaagcaagcaac ggtcagacaa 69480 cattaaaaaa atgttgatct gtaatgggtg gagaaagacctggcatgtat cagggacctg 69540 ggtggaaaat ccattaaaag aggcattatg aatagagtctgaagttctag agtaagaatt 69600 cctgttccac ttatatggcc taagttggta gtttggtgtttggatagggg agtggatgga 69660 agaaatattt tacagtttgt cagctaaaat tgtggtagtcactcactggg tacttaaagt 69720 gagatatact acccagagtt accttcagtg cgttctctcatctgtgcttt tatgatagta 69780 aagttttaag taagtttctt tgtgtttcaa gtaacagaccaacttgagct agtttaagtc 69840 atcatttaca aaaaataaac aacaacaaaa caaagaaacagaaagagctt agcgaaagca 69900 cccagagcct cttatagatt ccaagcaatt gaataataaggctttggaga aaagaatagg 69960 aaactgggca tttgtagacc agaggtcaca cagtctgtgtttcagtgttg taatgagaga 70020 atctcattgg cctggcttga tccaggcatc ttttcctactcaaataaacc atggacaggg 70080 ggctggagtc acattggatc atcagggctt cattgggagggggtgctgcc ttcttagttt 70140 gagggtaaag cagggttgtc agtcaagggt tagaaacatctcctaaaggt atccactcct 70200 ctgcagggaa tatggaaatc tttccctcat ttatggctaattaggcagta tagcctcatt 70260 aatcatggga tgacagtaca gtgtggtgga aagaggatgctgtggagcca cacaaacagg 70320 gttcagagcc cagctttcct gccctaatgg tggcactgtgcaggtcagcc ctgttgtctg 70380 taaaatgggt gtaacacaga ctaatgtgca gggtgggtgacagggttaga gaaaacttac 70440 atgacaggca aagcagcgta ctttgctcat agaactcaataaactgttcc tctgtaatta 70500 ttattaataa acattattgc agagtatggg cacagtggctcatacctgta atctcaacat 70560 tttgggaggc cgaggtggga cgattgcttg cgcctgggagtttgagacca gcctgggcaa 70620 catagcaaaa cctcgtccct acaaaaaata aataaaaattagccaggtaa ggtggtacac 70680 acctgtaatc gggaggctga ggtgggggga tagcttgagtcagggatatt gaggttgcag 70740 ggagctgtgc tcgtgtcatt gctctccagt ctgggtgacagatcaggacc ctatctctga 70800 aaaaaaagaa agaaaaacaa agtatgagtg aatcacaagtatggcatttt atcctggatg 70860 ttttgttttc ttatattctc attaccagtt tttatttttagtctttttaa actaccaaaa 70920 tattattctg tgatactcag aaagcatcac taaattcatacaaaaataaa aagtaaatat 70980 tgttaccatt atttgtaaag agatatttgt gaaatatattacaaatatat ttcacaaata 71040 tctctttaca aataatggta acaataatga ttgtatactgttaatataac caaactcttc 71100 cattcatctg ggcagtagag caaaaataag gcagcttacctcaggaggaa gttactagac 71160 tcaagattta tcctgaaatt tagcctacaa tactgacaggcagtgtcaag ctcacagtga 71220 tagatacaaa ttccaaaatc ctaattttga tttgaaagcttaagttttat ctttggcaaa 71280 aatactgtca atttgctttt cctcaaaatg acaggtttgctgctttcact taatatttgc 71340 taaatactca attctgatta accctggttt gcctgtcagttgctctttca attaaaatgg 71400 ggtttcatga agaaagcagc tatttcaact cacagctcaaacaatagcat tatagctctt 71460 cctggagaca actctcatac ttggcatgcc acagaaatgctttatgtata cttccatttc 71520 atcacatgga gtagtagaca ttactcaagg ttcgagatttattaaaatta taaagttact 71580 aattttaact tccaacaaag ccactcttga gtgaaactggcgtttttgct tgtttaactg 71640 tggcatgtgg aggtgagcag tagaataatt atagtacagtctggttctac tgccttgatt 71700 catgctgatg tgatcggttt caccccattg cctctgcaccatcagtttga atgttagcac 71760 agtgaaaagg aaagatcatg gctaagtatg attaataaaatcctttggac ctcctgtatc 71820 ttcctgaacg agtctgtgga ctgcattttg agaacttctgctttattctg tccagggttg 71880 gctggaattc accttagatt tcttaccacg ttgctgcatcacaagcagaa agccttagta 71940 tttctgtggt aacattgaat ggtttccgtt gggttttctcttccaggggt taattgtgaa 72000 attaattttg atgactgtgc aagtaaccct tgtatccatggaatctgtat ggatggcatt 72060 aatcgctaca gttgtgtctg ctcaccagga ttcacaggtaaagctccttt tactgcaagg 72120 cccctccttc agccctatca ctttgggaat atagcatccaaaggaatatt gcttttcaag 72180 tgtttcctat ttctttgtca ctctggaggg ttagtagtctgtttctctgc ttctctagat 72240 cagttttctg ttcacagacc ttttctaaac tgaaacatacttttcattaa ttagagtttt 72300 tcgattatgt gtttatttgc tgccagaaaa taattcctagacagaccata aattgccttt 72360 gctttgcttg agttacattg cttccaaagc tatgtttattgattacaggt agtagtcaag 72420 gccttccgta aagacatatt caaggtaaaa tcttatgaaaatactgaatg cttgcttctg 72480 ggtacctcct ctttgacata ctcaaggtag aattctgtgaaaatactgaa tgcttatttc 72540 tgggtacttc gtcttctgaa atattgttgg atgaggtcagagcttgggga gaccacccta 72600 atttgggtag catgagagag atctttctgc ccaggaggaagaaagtaaat ggctagaaga 72660 attttgaata taacttctct taaagtatga acagcctaggcatttacaca tacacacatg 72720 tgcgtgtaca tgcacacgca cacacagaca cacacacacacacacacaca cacatcttct 72780 ctctctttac ttgtcaatat tacctttatc ttttaccctcctgatcaaaa agtgctagag 72840 gctactactg cagaaacctg aggcccctca ctgacacacctgagtttctt cttggcatca 72900 tcagaaggca cacactgtac attctgttct tcctggacagtgacacacag aagcagccac 72960 taacatgtta ccctttaata atttccatgt ggttgtttgtactcgaatgt gagaaacacc 73020 cttcagagtt tttgatagag tctgcagaaa gcagccagagcaaagtgaag tgaatgtcca 73080 ccattcattt gccaagaatg tactttgttc tctatttcacaagaatttct cctatttggt 73140 atgattgtgg acattagaaa ggaaagtttg cacccaaatgggtcacagtg atccctgttc 73200 agtgaaagat cactgagaag caaaatctga tgaaaactgtcatacccaat gcccagtgct 73260 gctgtagtag cctgatgttc tgcatcctga aacttgttactccaagatca agtggcagtc 73320 gagtgaatac acaaaaatcc ctgctagata ggataataggtacattgttg ataagtgggt 73380 aatacaaaga gaataggtaa catttatgaa gaactaattttgagctaggc actaaattgt 73440 caattgtcac cgacgttgag aatacctaca gaataagaaacccagaacac agagatgtta 73500 agtaatgtta ccagtgcaca gcagccagaa ttacatcccaggcaatctga cccctgagcc 73560 catgctcatt cccactacac tggacatcct gattaggggcctgttgtgtg tgtgtgtgtg 73620 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtttatataaggc ttctactgga 73680 ctcatttttt tcagaatgcc aatgaatcat attctttctctttttaatat tcagcaaacc 73740 aggaaggcca agaagggaga acataggcta aaaatattctaaaataaacc taaggtgtag 73800 catgaaacct tataagcaag aaatggaatg ttttggttactaggtggtcc cctgttcatg 73860 gaactgttgc ctatgtctga gatgattttt ttaatcagctgctaaaaaca ttagagatga 73920 ctgtggtata tactcatgag tggtaaatgt gaggagttttattcttgctc ttgttttatt 73980 ttaatatatt tttcccatat cattctaata atagctttgacagctttaaa ggggattttt 74040 ttgttttaat actaagttat ctgttctctt agaacctttttaaatggggg aatgtaagga 74100 attatgtgat acttctataa actgcttttc agttttacaaagcaccttca tgcccattat 74160 cttcctgaat tctcataaca atccagaaag gaagttctactatacccgtt taacaaataa 74220 ggcaactgag agacagacct tgatgtatat actcatggcctctgccagtg agttgcatag 74280 tgaggactgt gagttcccag aaggcagaga cctcgtttaatcatctttat agcaagtgtc 74340 taaagcaatg tctggcacgt aggaaacgct tagtgaatgttgctgaatga acgaatgaat 74400 gggactcaaa tatagcgctt ttgctagcta atgccagactctgaagtgtg gactatacca 74460 tgttttcatt tattaaacac caatttattg agtgcctaagtcagaagaaa cactgggtaa 74520 cttctcattt tagttaccta gcccaaaaaa tttcttggccttccactgga ctttcctcat 74580 gctcagtgga aactccttga gggtaggacc ttcctgatggaacagctgtc aatcaacata 74640 gtggactgca tttaatgctt tcagataaag atggcttgaaaatttattct gcaaagaata 74700 aatttaaaag caaataaaga aaacttggga ggagtggcttctgaggagac gcttccaaga 74760 aaagaccaaa aatgaagaaa cctttttcct gttatccttcactatattac gggccgaggc 74820 taactgtgtg gttgaagagc tctggttcat attttggtgccactgacaaa cagctcaaag 74880 aatcagtggt tgaggccaag gaaatagtat ttaaaaataagctattaata aaacacttaa 74940 gaggaaaaca gcagccactt ccagattatg ggtttttaccacaagaacca ccaatgatgg 75000 tttagagagc aatggagggt gaaaaaatgt gaatactcatacttcatagg agtgggtatc 75060 tgtttcattt ggaatgtcct attctaaata caggattgctctttcttaaa tgagaaaaat 75120 gcatgttttc aaaatcagaa tcagaacatt gtgttctttgaaatatataa attcatagca 75180 gttgcttgat gccactcatt tatagaatta taggtaattttaacatttca tatcattttg 75240 gaatatatag ttaaatctca gttactcaaa atgtaacaaatggaaacact tgtataactg 75300 acccgtatta ctttattcag cctaagaaat atcaaggttatagcagacat ctttttaaag 75360 agcaatcttg gctatgtctt ggaggagcaa catattcacttcaatttcac ttactcccag 75420 tatccacatt gtatgatgat gataagtaat aattgtctagaacagaattt ctcaaaatct 75480 agtttttaca ccacctacat ttaaaaaaat atggtttgctagattctatt catcacaatc 75540 tatggtgata gggcctagga atctccactt aaaataacaacttaaagtga tgattttgca 75600 cattaaagtt taagaaccat aggtctgcag tgacagactgcattattttc tctaatatca 75660 gaagacaaca aaattatact ttgagactct acttcctgaggtcaaaaaaa atggattaat 75720 agactctaag aatgaaaaca aaataacctt tctttacaatcaatttaatt attgaattaa 75780 gttactagcc cattcattaa gcattccagt taatttggatttaccataaa tagaaatgta 75840 agaggctgag aaaatgaatt tgtgctaaac cggcacaaagcatgttaatg aatgcttctt 75900 tgagttgtac tcagggcagc attaagaaga gaagccttctttggtaacat ttgacattct 75960 cttcttagaa ccttgtgaag tttccccatt tctgagttgaaagaaaatgc caaattgcta 76020 taacactttt ccagcctttt gttaactttc cttgattatcaaaagtcaat gctctttgct 76080 cctttgcatg gcattaagtg ttattgggtg tctaattccttacatcttga catttggtgc 76140 ctcaaggcac cttatggcct gagcagatga aggatgtcaacataacgtgt cttatagttc 76200 tgggtctatt tcctaagcat ttaggatgtg actaacactgtcccccttct ccacagggca 76260 gagatgtaac attgacattg atgagtgtgc ctccaatccctgtcgcaagg gtgcaacatg 76320 tatcaacggt gtgaatggtt tccgctgtat atgccccgagggaccccatc accccagctg 76380 ctactcacag gtgaacgaat gcctgagcaa tccctgcatccatggaaact gtactggagg 76440 tctcagtggg tgagtagctg ccccatgtga tagtttcttattgacctgtg ttaagccagt 76500 tggcttgtag tactggtgac agcctgaagt tgtcaaatggggctttgagc tgaaactttg 76560 ctgaagattt ggtagtctgc atcaaagcct tctgaattaccatttaatcc atggaagctg 76620 gcccctccct tccataagtg aggacctaga tggaacataaggaggaatgt ctcccttata 76680 tgggtcatta gcaaggacct catgggagga acccatactcattgtatgaa tctttggcag 76740 ttatgttttt agtatgtatt actggaatga tttctgaagaatgttctagc ttttgatttt 76800 ttttcccctg cttaaacata gctcagctct gttctgtcacagtgccagtg tgtgctctga 76860 agtagggccg cttcagccaa ctttagaaat gtccctgataaaacatttgc aatttttaag 76920 ccccacagac ttgtgtgatg tgttagtgat tttctagaatcatagagtga ccttaggtct 76980 cacgagacgc tcttatactc ttgctgcatt tgccataggatatggctttg gacattttcc 77040 tttgccaaac tttgaaagat gaaaggataa agagtgtttagtggcaaata gtaaataaag 77100 ttatttaaaa attaattaac tcagtattgg tgtttcagagtatagcagcc ttcaaggcac 77160 ccttatccat ctctcatttt cttaaaattc tcccgataagaaagctatca tttcctgata 77220 tcacattcta agtgttagtg atgttcaaat tggagattatatttacaatt caaatacctt 77280 cattcctgtg ccttttcctg cagaataccc taggtgctttctgaagccaa gtgttaggtc 77340 aagacaaagc atgacttaga tatgaaatat cccaggagcatcttagagtg gctcagggtc 77400 cctgacagtg acctaggtat ctatttggca ccaaaaactaatgatcttaa tgtttgcttt 77460 actttagttt tatgtataat aacatacatt gtgcagatttatccatgata ttgtttcctt 77520 cagagtggcc caatatattg tagaaaccat tgtttacataagaaatttca ttataatttc 77580 actaacttag tgagccagga aagcaccaag aaggtttctttactggatac acctgatagg 77640 gccacatggt acacaccatg tgcaaggtga tgcaaggatagttccatgaa agtagaaatg 77700 agatcagttt accccatttc ttcaccaagt cctgtatttcactctatgac tcaatcatac 77760 gtttatacct cctatagtaa aaagttttgt cttctttcagatataagtgt ctctgtgatg 77820 caggctgggt tggcatcaac tgtgaagtgg acaaaaatgaatgcctttcg aatccatgcc 77880 agaatggagg aacttgtgac aatctggtga atggatacaggtgtacttgc aagaagggct 77940 ttaaaggtga aaacaaaagt acatctttct gcgtctgctctttgtcttct tgttgttgca 78000 tactgcttga ctcacttctt agtttccttt tctctttttcattcattcgt gtaacaaaca 78060 tattgagcaa ttagacttgt aaggcacttg gggaataaaaacatgaatag atacaatttc 78120 taccttcagc ctagcagaga ggtgacctat tgaaatagtaattttttttt tttttgacac 78180 agagttttgc tcttgttgcc caggctggag tgcaatggtgcgtgatcttg gctcactgca 78240 agctctacct cccgggttca agccattctc ctgcctcagcctccctagta gttgggatta 78300 caggcatatg ccaccacacc cggctaattt tgtatttttagtagagacgg ggtttctcca 78360 tgttggttag gctggtcatg aactcctgac ctcaggtgatctgcccacct cggcctctca 78420 aagtgctggg attacaggcg tgagccaccg cacccagcctgaaatggtaa tatttaagat 78480 gagcagttaa gactccagtt ttacagcatt ggccaagtgtctaattataa gcttcctggt 78540 ccaggtttga gtctatgata gttcaaatag atatatggaagtagaatgaa aatattatgc 78600 ttactataat taagatagta gtaatatcaa ggaagaatccacatgttcag aactctttca 78660 acaaactttt taacacattg agatattctt agttatttagtcatctttgg tctacaccgg 78720 aggtcctcaa ccctcaggct acagaccagt acctgtgtgtggcctgttag gaaccaggcc 78780 acacagcagg aggtgagcag ccagtgaggg agcaaagcttcatctgtatt tatagccact 78840 ccccgtcact cacattaccg cctgagctct gtctctgtcagatcagtggt ggccttagat 78900 tctcatagga ctgcgaaccc tattgtgaac tccacatgtgaaggatgtag attgtgcgct 78960 ccttataaga atctagtgcc tgatgatctg tcactgtctcccatcacctc cagatgggac 79020 tgtctagtgg caggaaaaca agctcagggc tcccaccaattctacattat agtaagttgt 79080 aaaactactt cattgtatat tacagtgtaa tattataataataaaagtaa agtgcacaat 79140 aaatgggatg tgcttgaatc atcatcctaa aatcatccccccaaccccgg atcatggaaa 79200 aattgtcttc cacgaaacca atccctggtg ccaaaaatgctgagggactg ctggcctacc 79260 caacaaatgg atagtcatgg gaattgtgta aaatctgcttctgagttatc actgttatat 79320 ttctaccatc agttttctct tggaaactca caaaagagagccgtgtgggt aaatcattat 79380 atttgcactc atctgtctat ttattcattg atttcctcactcaaatattg tgctatgcta 79440 ttgtgataca tcagcatgtt ttagagccac tttatttgattacttgaaac atttctgaga 79500 ctgtatttca gccattaatt ttctgatata ttgtattacattggcaaaga aacttgtaga 79560 tttcaaaaca cttttatttc tttatcttct gagacttgcttatcagtgaa caaaataaaa 79620 ttcaaaaggt aaaatcccaa attatatgcc cagtgggcagcagaatactc cttgaaccca 79680 tacctctgca ttcctagacc atgttttctt ttccacagtgccatagtttc tctactttaa 79740 ggctctgtgc caggccctat tggagatgca aaggtgtgcagtatttgggt actgtgctat 79800 ctccactaat tttttattcc ttttatcctt ttcttagtgggagaaggttt catgccttga 79860 atcaggagaa aacagctttg taacttcagg aagtaccacatgggtggtag acatggtgcc 79920 tatgaaaaaa tgggcataaa ccagaggctt tgattaatgtattcaaaaac atcatccctt 79980 ttcctgacca gaaatgttat atacaaagag cttatgtttatcatgcaaca agctattggc 80040 tgggtgcgat ggctcacacc tgtaatccca gcattttgggagggtaaggt gggaggatca 80100 cttggccaca gaaattggag accagcctag gcaacataacgagacctcat ctctacaaga 80160 aatttaaaaa atcaagtggg catgatggca tgcacctgtggttctagcta ctcaggaggc 80220 tgaggtggga ggattgctga gcccgagagg tcgaggctgcagtgagccat gattacacaa 80280 ctgcactcca gcctgaatga caagagtgag accccctgtctcaaaaataa ataaataata 80340 aatagatcct gcaacaagct tttgaaccaa gaacttcattattgagaagt tatttgtagt 80400 tggaggggca ttaggctttc agatcattag aatagactgtttttccattc atcttttcaa 80460 ctaattgctg tcatttactg actgtatttc agggacatggggctgaaatg aatgaacagg 80520 tatttcagtg gcctgaagaa atgaatgcaa tttctgatgtgttcagtttt tacccctaaa 80580 gagaattggt tagaaattga tgaggctgtc agaaagttttactgtttttg gttttttcct 80640 tttgtctaca ggctataact gccaggtgaa tattgatgaatgtgcctcaa atccatgcct 80700 gaaccaagga acctgctttg atgacataag tggctacacttgccactgtg tgctgccata 80760 cacaggtggg tcctggaggt accagcaggg accagaggggatacttgacc ttgctttgtc 80820 agtgcctgag tctcagtgcc caggtctgtg gggctgtaatgctcctccga gggctcactc 80880 ctcagttgtc cttcctgcct ttactgactg gatcttgagctttgaggaaa cattgtggcc 80940 tcctaagatg caagtggtgg acagtcattt gcctacattgtactttagta tttgaaagaa 81000 attttttttt aattgtatgt ggctggaacc aggcacatagatgatggttg tctatcattt 81060 gaatgtttcc aggacattag ctatttgtct tagtttcaataatacagcta ccacttattg 81120 gtaatttact atgtgccaga cactttttgt actttggattatttaatgta ttcctcaata 81180 attctacaat gtagaagttc ctattgccat tttacacatgaggaggctga ggcaaaggga 81240 gcttgcttgc tcaaggtcat ataggtaagt agtacatctaggatttacct gtaggtctct 81300 ttgaatctga agcttttcct cttaatggcc tttttacttcccacattcag taccgctaca 81360 tctcactcag aggtgtcacc ccttcgatgg ctgttacaactgagaagacg tgagtgtgac 81420 tgcatgtcac atagctgttc tttgtttcag ttgttatgtttggagtgtgc ttgggacatt 81480 tattctttgt tttctactgg cttattatcg ccttcttgtgattggttata tttctatggc 81540 ttgtttatct cctcttcatc tatttttctc attcagagggaacaaaacac aaacatagac 81600 aataactttg cctgtttcat caggttgcgg catgcagtcaggagcatggc tgagttatta 81660 cttccctgga gtcttgagct gttaatagca gctgttaatagtgactctaa gttgtgtaga 81720 gtagcagaat agccatagca tgactgtgtt acctgaaagacttgatagca cattgttact 81780 ttttcccctc tagaagaaat agtaggccag gcacggtggctcaccctgta atcccagcac 81840 tttgggaggc cgaggcaggc ggatcacgag gtcaggagatcacagccatc ctggctaaca 81900 tggtgaaacc ccgtctctac taaaaatata aaaaattagccgggcgtcgt ggtgggtgcc 81960 tgtagtccca gctactcggg aggcggaggc aggagaatggcatgaacctg ggaggtggag 82020 gttgcagtga gccgagatcg catcactgca ctccagcctgagagtcagag tgagactccg 82080 tctcaaaaaa aaaaaaaaaa aaaaagaaat agtagtgtagggaaactaca tttaacaggc 82140 ctagttcata aaatcacagt tgtaggaggt ccctaactatcttgcccaag gttacacagt 82200 taacaatatc agagccaatg ctggaaagat catgggcttctccattttca aagcctctta 82260 atcaggactt ttgacttgaa ttcagagaac atccttgggataaggaaaat tttcgttttc 82320 ttacctgttt atctgtcata aactctgtca tttactttctaggcaagaat tgtcagacag 82380 tattggctcc ctgttcccca aacccttgtg agaatgctgctgtttgcaaa gagtcaccaa 82440 attttgagag ttatacttgc ttgtgtgctc ctggctggcaaggtaagaac atgggtgtgg 82500 agaagccaag aacatgcatt ctgactttaa acaaagtggtcaggttatca gatcatatgg 82560 aaggccctgc tggtcttatg aggcctgtcc atcttgtcaagagcacacag gttttgcaaa 82620 ggagctgcta cttgaaatgg cttagaaagc ctttccattgtccttgtctt cggggccccc 82680 tagcggctgg aaggctgcaa cctaagcctc acaagccatgatgttcattc ttcagggagt 82740 ttaagcacct tggcagcaac ttcctaggcc acagaagtcttcctggaact tcctaaagca 82800 gggggccaac cagtttgtct gtggaatagc tccatgcaccccaactaagc tcaggtcctg 82860 cgtagatttt tgtgggaatc agcttccaag gactaggtcatttattggcc ctgtgttcct 82920 aggtcagcgg tgtaccattg acattgacga gtgtatctccaagccctgca tgaaccatgg 82980 tctctgccat aacacccagg gcagctacat gtgtgaatgtccaccaggct tcagtggtat 83040 ggactgtgag gaggacattg atgactgcct tgccagtgagtatgccagtc agctcttaag 83100 cccccagagg agaggaaaac acacagggag cagaggagagcccagtggag gctgccgcct 83160 ttcacgcctg acattgggca taccggtcat ttatccagcatatttaacag tagtgtttta 83220 actctttctt ttgaagctag tgtccttggg aattgaggcagtggaatata ttttaagcat 83280 tccctttaat ttgtgaacat tgaaaatgtg aggacaatgctatgatagtg ttttttaaaa 83340 aacagattta ttgaggtata attaaggtac attaaactgcaaatattttc agtgtacaat 83400 ttggcacatt ttgacatata gtaagtgaaa ctatcacccaaaataataaa catatttgtc 83460 accctaaaac tatcttcatc ccattgcaat ttaaccttctctctttcttg cctgtggagt 83520 cacagtttta ccttctgtca ctatagatta ttagttggcagattttatca ttttatataa 83580 atgcagttag ataagcataa gtcaggagag gttctgacttgttttaatga acatagtatt 83640 tgagatccgt ccatgttgtt tatatcaata gtacattctagtttattgtt ctgcagtatt 83700 ccattgtatg gacatattac agttcattgg ttgatggccatttgggtgtt ttccaatttg 83760 gggatattac aaatgaagtt gctatgaatg agtctttgtgtggacatatg gttttagttc 83820 tcttgagtaa atacctagga gtagaatcgg tgaatcatgtcgtaagtgta tgtttcactt 83880 tttaagaaat tgccagaatg ttttccaagg tggttgtactattttcattc tcaccagaat 83940 tgtatgaaag ttctgtttga accatatgct cacaaactcttggtgatatc atttaaattt 84000 agctcttcta gtgggtgtag agtggtattt cattgtgattttaatttgta ttattctctc 84060 tgccaatgat gttaagcatc atttcgtgtg ctttgcccatctgcctatct tcgttggtga 84120 aatgtctatt taaatttttt gcccatttga aataattaggttgtcttctt gagttgaaag 84180 agttctttgt atttttaaat aagaattctt tgacaggtatttgcttccca atgatttccc 84240 tccaatttat agcttctctt ttgattttct tttgaagaggaaaatgttaa cattgatgaa 84300 attcagtgta tgcatgtttt tcctttatag ttcatgcttcatgtatcttg tttaacaaat 84360 cttcacctaa tccaaatttg aggtttatct actgtggtttcttctaaaag ttgtgtaatt 84420 ttagctctta cattttggcc tatgatttgt tttgagttaatttttacata ttgtgtgact 84480 taaaagtcaa gtttaatttt gttttcttac tgtactaaactaacttctta ttagttgtag 84540 gttcgtaggt tagcctctag atagtttaga ttcttaggattttctacaga tacaatcatc 84600 ctgtctataa ataaccctta tgccttttaa tttcttttcaaccctgtatg ccttttattt 84660 attttagtat tattgtacta agatcttcaa taaatacagaatagaagtgg tgagagaagc 84720 cttaccttgt ttttgatcct aggggaaaag tatgtagtcttttaccatta agtgagatgt 84780 tacctatagg ttttcataaa ggccctttat caaattgagagaccttgtac ttctggtttt 84840 taagaggtct ttttgtattt ttataatgaa tgaatgttgattttttggtg tgcaattgat 84900 ttctctacat ctgttgacat ggtgatatta ttttttggtcttttaatgta agtaaattgt 84960 catcaacttt atttaacatt ttgcaaatgt taaaagctaatcttaaattt atgggctata 85020 ttccacttgg tcattatgtg ttattccttt tatgtattattggatttgat ttgccaaaat 85080 gttgttaagg atttctatat ctgtgttcat gaaggatattgttctccagg tttcttggac 85140 taccattgtc tggttgtgat aagagggcaa tgctgtcctcatagaacata tcaggaagtg 85200 tttttctttc tactttctgg gagagtttgt gtagaactggtgtgttttct tccaaatata 85260 tagacatgga accttctttc tgggaaagtt tttaactacaagtttggttt tcctaataca 85320 tataagtgcc atatgggcta tctgttttta attgagctttggtaatatct gtctttcaaa 85380 gaattagtcc aattcacctg ttttgtcaaa tttgttgacataacatcaat ttgttttata 85440 acatttcctt actgtgcttt aatagtctat tagatctgtgatggtttcct tctcttgttt 85500 ctactattgg taatttgtct cttctttctt tgaaagggaagaatgtctgt tttgcagatt 85560 tttttcatac agtgttctat aaatgttatt aattcaaattggatagggta gtgttattca 85620 ggtcttttat gtcctctatg actttctgtc tacttattattcatagaata taataaggag 85680 gctttctgtt tacttcttat tcaagagtat tgaaatcttttactatcatt gtatattcat 85740 ctgtttctct agtaattcta tttgttttgg cttaatgaattttgaaactc taatcttaag 85800 tgcataaaca tttagggttc ttatgtcctt ttgaggaattgacccattta tcattttgaa 85860 atggcctttt taaactcttg tcatatttct ctctctgaagtctactttgc ctgatactga 85920 tatagctact ctagctttat tttattatta gagtgtgtttttaaaaatcc ttttacttct 85980 ggcctgtttc tctaaaatgg gtttcttgta gagagcgtatattgggtctt gtcttttttt 86040 ctccagtatg atggtgtcta tcttttaatt caggtatttagactgtttaa atttaatgta 86100 gtttttatat ggctggcttc aaaatatgcc atcttgctgtttgctttatc tttgttcttc 86160 tgtgtattgt ttccattttc ctttttctga catcttttgaaataattgag tatttttatg 86220 attacatttt atctacatac ataaatttca aaactttatctatattcact accatattgg 86280 ttacatattt tttagtagtt attgtagggt ttactggtgtgcagctttaa cttattataa 86340 tgtaccttca atcgtaccac ttcatataga ggagaagagctgtacactca catttacatt 86400 cctcctggcc tctcttctat tgttgttata aaatgtatatagcattaaac ccatcaaata 86460 tgtaattaaa aggtcaattt ttttcaagat atttgaaaaataatttaaaa gtctttaagt 86520 tacctacgta ttttcaagtc ttattggtct ttattcttttatgtagattt gtgtttccat 86580 caggtatcat tttccttctg tctgaaaaaa tcccttgaatattttgtgta gtctttgtct 86640 cctggtgatg aagtctttct gtttttgtgt gtcagaaaggtttttatttt gctttagttt 86700 tcaaagatat tttcacaggg aggagaattc taaattgactttctctttca gcactttaaa 86760 gctatcctat tatcttcttc cttgcattat ttctgaggagaaatctgctg ttattccaca 86820 attcttttat ctgttccttc atatataatt ctctctcctgtggctgcttt taagatatcc 86880 tctttccact gatttttacc aacttgatta taatgcctgttggtgtggtg gtcttttttt 86940 tttttttttt tttttagact gagtctcgct ctgtcgcccaggctggagtg cagtggcgtg 87000 aacttggctc aatgcaagct ccgcctcctg ggtttgcgccattcttctgc ctcagcctgc 87060 caagtagctg ggactacagg tgcccgccac cacgcccggctgattttttg tatttttagt 87120 agagatgggg tttcaccgtg ttagccaggg tggtcttgatctcctgatct tgttatccgt 87180 ccacctggac ctcccaaagt gctgggatta cagcgtgagccaccatgcct agcctggtgt 87240 agtgttcttt atgtttcttc tgtttggagt ttactgatatttttggttat gggtccaagt 87300 ctggaaaata ttttaaccat tatttggtaa atatttagccattatttttt tcaaaagttt 87360 ttgcgtctcc cttttataat gttcattatg catttattaggctgcaaggt tgatttattt 87420 atttttcagt gttttttcca cttagtgttt tattttggattattttacta tttgaagtta 87480 ttttgttata ctttcaagtt tactaattgt ttcctctatagtttctcgtt tactgttcat 87540 ctcatttagt atatttttcc ctcagatatt atgtttttatatgtggaagt ttaattcaag 87600 tctttctttt aacgttacct ccctctttgt catgttcatgcttttatttc cttcttgaat 87660 atattgagta tcgttgtttt ctagttgata ataattgaccatattttatt gtccttacct 87720 acttttctac catatgagaa attcctgagt atgtttctgttcatattttt tctactcatt 87780 ttgaatgtat ttcctgcttc tttgcctgcc tggtaactttttactggatg ctagacatcc 87840 atcttacatt gttccatgct gaattttttt aaaaatcttttttaaaaaaa catttttgag 87900 ctttgttcaa atattcaatt atttggacat agttcaatccttcagagtct tacttcaagg 87960 tttgttaatt ggttccaaaa tagtctttag ttgagggctaatatggctca ctactacaac 88020 aggggtccct caggactcta cttactgcca cgtggtttttttcattctta ttgaataaac 88080 atgagctgtt ctcagcccag tgaaacctct agggattattctgcctactt ctttctggtg 88140 gttcttcccc cagccttaat agttttatta catgtatgtactgaccgatc cttagttact 88200 tgtatagtga tttaacttca aatgagatta tcacaatgcattaataatgg caccttaatt 88260 tacaggatat ataacttcag attatcatat agaggccaccatgctaggtg ccggcagtga 88320 taagataaat aagttttagt ttctattttc atgaagctcatagtctgtta gggaagaaag 88380 acatatagac acagttcaag accagcctgg ccacatggtgaaacctggtc tgtactaaaa 88440 atacaaaaat tagccaggcg tggtggcagg cacctgtaattccagctact cgggaggctg 88500 atgcacgaga atctcttgaa cccgggaggc ggaggttgcagtgagccaag attgtgccac 88560 tgcactccag ccggggcaac agagtgagac ttcaccaaaaaaaaaaaaaa aaaaagacat 88620 atggacaaat aattttaatg atgtattaca aatagtgaaaaagtatccaa ggcacaattg 88680 atggagaact ttagggagtg agatcaagct gtttgtattcctttctcaca ttaagtaaca 88740 tgtcattcaa aatattgagt atttttaaat ttttctgtatgaagtgggac caacaatatt 88800 tacaatgagt ttaaaataat tcagtaaatt atggatggtgagtttcctga aagatgctat 88860 ggaaattata gtagctgtgt actgtggtta agaaaaactgaaacaatttt tacaattgct 88920 ttaatagaga ttgcagatga gtcttataag ggaattagttcaagagatga ataaagtata 88980 ttaaatgttt cgctaataaa ataaaggtca cattaccctttagtttagta tttagttttc 89040 attttacatg tatgacctgt ctttgcaatt atatttttaagttccttaag agccatagct 89100 acagcttatt tttcttgttc tgatttgtat ggcatataattgtagagtgc cgtggcatga 89160 ctgtagctta aaatttgttg gttccttact atgagtataaagtgttccaa gatcaagcta 89220 tgtgatattt caatttttat ttaaactgtg tttcataaggctactgaaat ctaaaagaga 89280 gagaaaaaga gagagagagg agatggaaat gaaagggagagagaagatta tataattaat 89340 atgcctggaa aaacatgaat atgtgtattt tgtcctcccccacacactcc acaatcacat 89400 ataccttttg aaaatattga ttgatattct gtgtattggggcacatattt gatttccaaa 89460 tgtgtgaggg accaatgcaa gttttatgac tattaattttacaaaactgt tggtggtgac 89520 atagatgaag tttattccaa ttaataaaca ggttaaaaacttcaagcaag tattattgaa 89580 ttcattttca taaatgaggg agttggcaaa attgaatgaaaaagtcagaa ttatgctgcc 89640 cccagcatga cttagttcag gtcattcact tttatgaatctaagattaaa ctgatgctac 89700 tcttcagagg tctaattctt ttgtaatggc ttagatccttgccagaatgg aggttcctgt 89760 atggatggag tgaatacttt ctcctgcctc tgccttccgggtttcactgg ggataagtgc 89820 cagacagaca tgaatgagtg tctgagtgaa ccctgtaagaatggagggac ctgctctgac 89880 tacgtcaaca gttacacttg caagtgccag gcaggatttgatggagtcca ttgtgagaac 89940 aacatcaatg agtgcactga gaggtgagca accagtccctcgggcatcag catcttacga 90000 gagaaaggga gaaagggaag gaaatttcag agcacaatgctagaattgtg gagcagggat 90060 gaggtgtcca catggatccc agtccacagt catctcctattgcccggtta ctggttcctg 90120 ataaacatat ataatagaaa gttaaaggtc agaggttgcaaagtttatag ggatggttta 90180 ttatttcttg cttagggaac agaaagagct ataattttgttctgttcttc ccttggcctc 90240 tcattcattt caaaaatcca aaaattccag tttttctgcagagattggaa ttgttactag 90300 tctttaagta atttctggga gttttgtttc atttctttttataatattat gagcatgttt 90360 gttctatttg tcttttataa ataatctata gtgaataggacttttcaaat aaattaaatt 90420 gacttagaga taaaatgaaa tgtgatacat gggaaaactacagaaggact gggtaaaacc 90480 tagaaaactt cattttctgt aggtatctat agtaagctcagtcttttaaa tacttctaag 90540 cctttgatta tagaaaggaa gtgggcacct cagagcttctggcacccaat ctagcttgta 90600 agaaagttta agggatatgg gttgctcaga ggtatttgcctctgtatgat cttagattcc 90660 agaaatctta ctttgtagta gtaagtcaca ggcaccagatacatagctga tgttgctctg 90720 tttgcttctt agctcctgtt tcaatggtgg cacatgtgttgatgggatta actccttctc 90780 ttgcttgtgc cctgtgggtt tcactggatc cttctgcctccatgagatca atgaatgcag 90840 ctctcatcca tgcctgaatg agggaacgtg tgttgatggcctgggtacct accgctgcag 90900 ctgccccctg ggctacactg ggaaaaactg tcaggtaattgacttccttc ccattcactc 90960 agctccccta gagcagctga atattatacc tgttcactttctagtctgaa taagttcctt 91020 ctaaaagaga aaattgtgga tctcagggat tccctgagataatctaggta acttcttaat 91080 aacttgcaga ttccaaaatg gtgctggcaa tatgatatagattcacaaac ataagatctt 91140 gaaaagaggc taaccaagaa ctgcagaaaa atacatgtgggaaataaaca cagatttttg 91200 ttttttgttt tttgtttttt tgttttttac attggaagcacaaaaagaac tgaagaaaat 91260 agagaataat caaaataacc ttttaaaggt cagatgcaacatctaagtgt ttaataatga 91320 taattgttgt atcttgcaat gtggtaacat tctcaaaaccaaacaattta catatctttt 91380 aagttcaaat atgtaaatac acacttccat taaattttagaatggaaaga gcccttatag 91440 gcaactaatc taaccttcga tttcatggaa ggcgaaacagccccagaggg gcaaagtaac 91500 ttttccaaag ttgcatacca aacagccctg tgtttaatttaatgttattc tacctcttta 91560 atagtgatag aataataact ccaaagaaca taggaataaatggccaaact taacttgact 91620 tacagggcgc acttaagtaa ggcttaggct atattagacatgtgagcacc cacctccaca 91680 aaattaagca gcttgttgct gattccatgg atcacgagagcatttttggt tgggctccta 91740 tttgttctca cagtgtagat tggcagtgct ggtgaagcactcctcccttc ctccagcaaa 91800 ctgtctatat tgtgaatttg gccttcctct cctcaaggggccaagccact ccgtgtgtga 91860 attaagatga aaggtgccag tccttgtgca ctctttgcttcagatttttc ataagtctga 91920 ttgccttcct tagaaataag ctcttaaacg gttttaggcttattaacagg cattaagtcc 91980 ttttacccat attcaccaaa gatagatttg gtcaagacccatgtggattt ctcacacagg 92040 attagcacag aaagtatgaa cctgtctgac agctttaactctttctttct agcattacag 92100 ggtggctata catgtaggca aatcatgtct agagcattgggaatagctta acctgttggg 92160 tttccctgaa tttttaaact tttttggtaa gaactttttaaatagaagac ctaagaggaa 92220 ttagactaca gcaggccctc aagtaacttt gtttccttcggtgtcatttt gttacaacat 92280 tgagtggggg aaaaatctgt ttttggccag agctgctatctgtgtggggt ttgcatgttc 92340 tccccatgtc tgtgtgggtt ttctccaggg actccagtttctttccacaa ctcaaagtca 92400 tgtatgttgg gtgaagtgga catgtctaca ttgtcccattgtgccagaat aggctctggc 92460 cactcatgaa cctgaactgg agtaagtgag ttagaaaataaatgaatggg gccgggcacg 92520 gtggctcaca cctgtaatcc cagcactttg ggaggccgaggcaggtggat cacgaggtca 92580 ggagatcgag accgtcctgg ctaacacggt gaaaccccgcctctactaaa aatacaaaaa 92640 attagccggg tgtggtagcg ggtgcctgta gtcccagctactcgggaggc tgaggcagga 92700 gaatggcgtg aacctgggaa gcagagcttg cagttagcggagatagcgcc actgcactcc 92760 agcctgggca acagagtgag actccgtctc aaaaaaaaaaaagaatgaat ataccaatta 92820 ttataaaatg aaaacccata aagcctatga taatcatatgagtgcacaac aataaacaat 92880 gtggtacaaa accacccagc aagcctgcca tattggttagtgttgctttc tgagctgtgt 92940 ggtggtaaga ggtgctgacg gttttgcttt gcaaacatttattccttgac ttgacctgcc 93000 accactagga ctgctattac tcatggattc accaaaaattggataaataa ttatcttgtt 93060 tttattaata tttcttaaat gaatgtatag cttgtttattttaatgttta atatcagaag 93120 tgggtgttaa tttgaagttt agtgattttt tttttaaccagaaataagta gaggaactta 93180 actctttttt atatcaatta gactgtggta aaattggtttcgttatatat cgttgtcctt 93240 aaagtcacag tttccaagaa cctaatgatg acattaagtgaagacttaac ggtacgtaaa 93300 aatggttagg tgtacctcgg aacactgaat ctagtttaagggctctttta ctagatgaag 93360 gctctttttc tttttaatca gtttgctttt ctttgatttcttgtaacaca atataacttc 93420 ttttcttcat ttctagtatt tgaattttga tgtatgtacacccctgcctc tgtttcagac 93480 cctggtgaat ctctgcagtc ggtctccatg taaaaacaaaggtacttgcg ttcagaaaaa 93540 agcagagtcc cagtgcctat gtccatctgg atgggctggtgcctattgtg acgtgcccaa 93600 tgtctcttgt gacatagcag cctccaggag aggtaagctttgctcccttg ggctcaggtc 93660 attcactgca gtattgactg tccagtggca tttagagaaggtatcatttt tttgtggaat 93720 aagagggaag ggcagatagt agtgggtggg cagggctctggggaaagggc tcaagacaga 93780 aggtgtaagg gtagaggaat aggttcagag attcggagaagaatgtattt gaaatgaaac 93840 taaggtatat tccactttca gggtttattt ttaatatgcatctggtactg agggttgtga 93900 agaactggct aagaataaga ttggatgtgt cagccagtttagatttactt gctcattggt 93960 tgatttgaac cccatgtgtc acatgttctt ctatgacatttctccctcac tgggtttccc 94020 aggtgtgctt gttgaacact tgtgccagca ctcaggtgtctgcatcaatg ctggcaacac 94080 gcattactgt cagtgccccc tgggctatac tgggagctactgtgaggagc aactcgatga 94140 gtgtgcgtcc aacccctgcc agcacggggc aacatgcagtgacttcattg gtggatacag 94200 atgcgaggtg aggacactga gcaagcagag gtcatggacctcccaggaga ttgtacaact 94260 gtttgagcac tgatatattg aatttagctg cttgtatcttatattgatag tttatagatt 94320 tttaccaatc cccgtcataa tgtttaccac tgtttctttcagcctctctg tgcaataagt 94380 taacaggtcc ctagtatgat atttttatag gcgtctggaaccatcccagc tccttaggct 94440 tgcttttagg attcagagcc aagagtcact accagagtgcattttacagg cccacccaac 94500 atagcagtcc acacttccct gaatagcagc tgagataggaaaaactgctt tgtggagtgt 94560 ccctttggcc cctgtcacct ttgtatttag tcattaatcagtcaactagc aattttctct 94620 caaaaaaaaa aaatgttgct catcctgttc cccatagcatcatgctaaac gttacatgta 94680 tatatttgag gaagtatgtg tcttcaaggt acatgtcactattttaatgt ctgggctgat 94740 aatggttaga gcaagcaaga tattagcaaa atgcctgagacattaatttg ataccaattc 94800 aaaccatcaa gcaacactga gaaaagcttt gcttctctattgttatataa atgagccaac 94860 gatagcttct gtattttggc ccctaggttg tttatttcttcacagcaggc tggggctggg 94920 tctcattagc tcaaaagcct accagtgcca aacccaaattttgacacatc tagttgtttt 94980 aaacatagcc cccaaataag cagattttta gctatttagagcttgcctgc tttgcatagc 95040 ctgtaaaact gcagccaaaa tctgctagcc atagtataagtaggacaaga tataccctgg 95100 ggccatacag accccaggct gatgctggcc tttagagctctctgacccag agattcctgc 95160 cactggctgt tgagtgacat cacctagact ccttctctgatccttctttc ccagagttta 95220 cttgctgtct tccccttact cacctacccc tctggaaggtctcatgctat ttgggacttc 95280 tcccaggtgc aaacctgtca aagtgtccaa ataaagccacctcacagcca tatctttttt 95340 cttggtcagc cctgaaatcc ttccaacccc tacagccaccacagccagcc agccagcccc 95400 acctccctct acccctaagc ctatctcaac cagtttctcacacatagatc caatttccta 95460 aacctggaga tcaataattg catgtctgta ctaccatccacatatgcctt ttgaccgagt 95520 catagggtcc tcatcacata cggaggaatg tgtattttaaagatgaggaa acctgtaccc 95580 aggaggaaca aaaggtttgc acacaagtca aatctgaaacatccttcatt gtattcagtc 95640 cttgccttcc ctttaaagtg cctttttcaa ctgaaagaggagtaggtaaa tgccagtaag 95700 ctgcaggttt tctgtccctg catagaatga ttagcagagagtgtgatggt tccagagaaa 95760 acatacaaaa gctaaattag gaggtgggag acagtcacagaaatgttcca gatctcacta 95820 gctctcatgg tctgtttcca gtaatggtcc aatatttcttgtcctttttg tttcctcatg 95880 gcagtgtgtc ccaggctatc agggtgtcaa ctgtgagtatgaagtggatg agtgccagaa 95940 tcagccctgc cagaatggag gcacctgtat tgaccttgtgaaccatttca agtgctcttg 96000 cccaccaggc actcggggta tgaaatcatc cttatccattttccatccag ggcattgtct 96060 taagttataa atccattctt agtgttcagg ggattttataaaattaaaga taggaagact 96120 agcttcattc caagcattta gttctacatc ctagtaattcaagccatttt attctcccat 96180 ctcttgctag ctctgatgtt gtggtttatg ttgtcagttttatctggttg tttggcatct 96240 tgatattcca tgaaacacag aatatggaag ggatacaacattagcataac attaaaaaat 96300 tagcctggtc agtaagattt cttgttgctt cacagaaaagcaactaatgg cctctaaaat 96360 aaacaattta catttacctg cttttgtatt tttttaattagccaagtctt acctctataa 96420 ggtgggggaa tgaatgaaat gaaaatatct gcctattttcatagcttcag gggaggatac 96480 tgatggtgta aggttctacc ttaggatgca gtaaggcctgatgagagatg caaccctgga 96540 ctgttgagtg atggggccat cattcttctg tgggcattgccactgctttt gaactgagtg 96600 gtatggccat cattctgctg tggacattga cactgcttttgaaccatccc ttggaaagag 96660 ctttgagtat tgtttgttag aagggatgta gttatgctggggcctactct gagaaggctg 96720 tgaaaaaaat gtaaaaggcc atccacactg gtgagaaattcatccttgtt agtgtcaaag 96780 agaatagaat gccagagaag tatttgtatt gccttagagctttgtgtcag taaagtttga 96840 taatttgcca ccccaggtat cagagtttga ctaaattaaaataggagagt accctagtag 96900 taggctgatt attaaggaat gaggttaatg ggaagaagaaaactaaggag aaacagtctt 96960 taagtatttg aagaattgtc ctgtaaaaag gagaattgttctttattcta gaaatcagtg 97020 gttgagtaga aattaattcc agttcattgt tagtttgttcctttgagaaa ctgtactggt 97080 taggacaagc tacgattctg gtgatagctg ttatcgtagttcatttatgt tgctataaaa 97140 gaatacctga gactaagtaa tttatttttc gaaagaggtatatttggctc gcagttctat 97200 aggctgtaca ggaagcacgg tgccagcatc tgcctctggtgtgagcgtca ggctgtttcc 97260 actcatggtg aaaagtgaag aagatccagg gacccaaacacctcccatta ggccccacct 97320 ccaacattgg agatcacatt ttaacatgtg gtttggtgcagtcaaacttc aagctatagc 97380 agctaattaa acctcaaatc tcagagctta atataacactgattttgtca ctcaaataaa 97440 gtcgaatatg tgttgggcta cctttctctg tgttttagatatgtggtttg aaacacctgg 97500 cctccaaggc ctccacagta cagggaagag ggacagacttgaagggtcat acagcagctt 97560 ccactagctc agtctggaag caacacatat cactatccctcgcagtatat tgaccggaat 97620 tattaaatgg tctctgtgac ttcaagggag gctgggaaatataggagagt acatgagtat 97680 tcatcaaact ctaaaatcac tactatacca tgaacttttaattaataaga catgtccagt 97740 cctagaatgg atcatttcag aaggaaacaa aatttttgtctcttctaacc atatggtaaa 97800 gatggcttag aatactagtt ttgatattat cattgacagtctttttcttt cagcactttg 97860 aatatatcat cccactgctt tctgatatcc atggtttttaatgagaaata tgctgttaat 97920 cttattgagg ttcccttata tgtgatacat tgcttctttcttgatgcctt caagattctt 97980 tatcttttga tagtttgatt atgatgtatg taggtatgaatttctttgag tttatcctac 98040 ttacagttcc ttggattatt ttactgtgtg tgcttgtttaggtatgtgga acagtatttt 98100 tttatcctga cttaacttct ctggccattg gtaaacattccaggaaactc tcaagtccca 98160 taaccccatt gttagagtca tcacaatgtg tgttttggtcaccatatttc taagccttag 98220 atctttgcct gcatgtccca ccagtttccc aggtcaagggctaaatgtaa cagttctgcc 98280 tcttcctact actttgcttc aggctgaggt tgagttctctctcagttgat agatctgttt 98340 atttctggtg gttccattct gatggggaaa tatgcatttatgctcatttt actagcaatc 98400 cctaaatatc aaatattctt tgagacaggg agcatatactagattttgtt tgtttttact 98460 tttttgtttt caatttactc tatgtataag gtgatatatgcatatatgca tatagaccaa 98520 aagtgtattc acacatagaa aagtatacag tgaaaatacttctcattccc atctctcttc 98580 tggtgggcaa attttccctc tcctgcaaac atgtaatcgccacttttggt tgtttgtgtt 98640 tctttctaga atttttcaaa aactgtatcc ttcagaatttctttatgtag agagaagcca 98700 aatgcaaaga tattttattt cctttacttt ttactctgaagatagaatat tagacacatt 98760 ttttgcactt gatttttttt tacttaattg gagatattttcatatcaaaa tatagaactt 98820 cctctctctc tctctgagat agatagatag atagatagatagatagatag atagatagat 98880 atttgtttgt ttgtttgttt tgagatggag ttttgctcttgttgcccagg ctggagtgca 98940 atggtatgat ctcagctcac cgcaacctcc gcctcctgggttcgagtgat tttcctgcct 99000 cagcgccctg agtagctggg attacaggca tgtgccaccatgcctggctg attttgtatt 99060 tttaatagag acagggtttc tccatgttgg tcaggctggtcttgaactcc tggcctcaag 99120 caatccaccc gcctcagcct cccaaagtgc tgggatgacaggcgtgagcc accacaccca 99180 gcctgaactt cctcaatatt ttttatagct acagaattcccttttacagg cataccatat 99240 tttaaccagt gccatcatga tagatatttc agttatcttgcaggcatata agtatattgg 99300 tgaaataaat tttcagaaat agaatcgctg gattggataatatgtgcatt tatcatttta 99360 atcagtgatt gcagtatatc agcatattgg ctttttcttaaggtggagaa tatattttca 99420 tttaagaatc attttctttt gatttttttt gcagtttctttgtttatata tatatatatg 99480 tatatataca tatatatata tatacacaca tatatcttttttcccacatt aagttgttga 99540 tgtaatgttt tgtggaaatt ggctatgata taaaaaaacaaatatattcc cagtattttt 99600 ttatcttttt tctttatagt gatttttagt gcacacttttactttttaat caagttgaat 99660 ttatcacttt taaaatggct tctggatttt gagttgtagttagcaacatc atatacactt 99720 gaagatcgta aagaaattct cccatatttt cttcaagtacttttgccata tgactactca 99780 tttgttaata cacaatttat tgaatatttt gttttttatatatataatga tttgagatgg 99840 cacctttgcc agattctaaa ttcccatgtg tatttttggtgttcttctgg attttcttta 99900 ttttctgtgt tattctatta attcaccaat aacgttctatttcaattatt aaggatcttc 99960 ttcttcatag ttttcctggt tatttttttt cacataattttataatcagc ttgtctgtta 100020 ataaattcct gttgataatt tttagtgggg tctcattaaatttatgaatt atagtataga 100080 gaaaactgat aactttatga tgaggagtct tgctaaccaaatcctggtat gtctttccat 100140 ttattcaagt ctcttttgtg tctgttagga cagtattaaagttgtcttca tttaggtgta 100200 acacatttct tgtgaagatt gttcctaggt attttatctttggtaatttg gtaattgctt 100260 ttgaaaattg ggccttactt tacatttaaa aaaaaaaatcttgcttttgt tagtgtatgc 100320 tacaatgcaa gtggcaggaa tacatacaga actgttttcttttgtacctt tttttttttt 100380 ttttttaaga cagagtcttg ctctgttgcc caggctggagtgcagtagcg caatctcggc 100440 ttactgcaag ctccacctcc caggttcatg ccattctcctgcctcagcct cccgagtagc 100500 tgggactaca agcgcccgcc accatgcctg gctaattttttttttgtatt ttttagtgga 100560 gacggggttt caccatgtta gccaggatgg tctcgatctcctgacctcgt gatccacctg 100620 ccttggcctc ccagagtgct gggattacag gcttgagccactgcacccag cctgtacctt 100680 tttttttaat tatgattatt attatttttt gagacagagtcttgctctgt tgcccaggct 100740 ggagtgcagt ggcataacct cggctcactg caagcttcatctcccggttt caagtgattc 100800 tcctgcctca gcctcccaag tagctgagat tacagacatgtaccaccaca cgcagctaat 100860 ttttgtatat ttagtagaga tggggtttca ctgtgttgggcagattggtc ttgaactcct 100920 gacctcaagt gatccgcctg ccttagcctc ccaaagtgctgggattacag gtgtgagcca 100980 ctgtgcccag cctcttttgt gcctttttga attaatcatagaataatttc tagaatagtc 101040 attgatttct tttgtttctt tgtaagacac tatatgcagatattcatact agatggctca 101100 tttacatcta tagtacaata aattagtcct gttttaaatggaataacttt tgaactctca 101160 gcaggaaatt tatcctctat ttttgttaaa taaaaagcattggtgcactt atacactatt 101220 acagaaaaaa gccagtgatt ataaaactta aaaatgtatttcattcaaaa ttcagttcca 101280 gttgttacca atatttttac ataacctatg tatagtcatgggtcacttaa cagtggggat 101340 atgttctgag aaatatgccc ttaggcaatt tcattgttgtacaatcatca tggagtgtaa 101400 ttacacaaac agagagagta tggcttacta cacacctaggctccaaacct gcaccttgtt 101460 ttactgaact gaatattgta ggcaattata acacaatggtaagtatttat gtgtctgaac 101520 atatttaaga agaggtacag tagaagattt aaaaggtacagtaaaaatac aatataaaag 101580 ataaaaaata gtacacctgt atagggcatt tactatgaaaggagcttgca agactggaac 101640 ttgctgggtg agtcagtgaa tggtaagtga atgtgaaggcctaggacatt actgtacact 101700 actgcagact ttgtaaatac tgtacaccta agctacactgaatttattta aaaagtaatt 101760 gtgctatgac attacaaggc tatgatgtta ctaggcaacaggaatttgtc agcttcagct 101820 tcattatggg accactgtcg tatgtgctgc ctgcattgactaaaatgtca ttatgcggtt 101880 gcataactgt atttattgtt atgttctttt ttggactgtttttatatttt ggattatact 101940 ttggaaaaat tactgagcct ctcttgagct ccagtttccttctctttgag atgggacaaa 102000 taataggacc tacaccatag ggtgctactt cactggattgtgtgggagag tacatgcgga 102060 gccctagtat gttctttcct ttacttgtac aactcaccaggatgacagct gtcatgaata 102120 aagagtgtgc tttgccctgc ccaactacaa ggtcccactaccagttgagg agtatactct 102180 gattcattgt gaaaatgcca acaagggtgg ttatcccttgctggaaagca ggttaaaagg 102240 attttcataa ttctgtacct cttctctttc ctccaggcctactctgtgaa gagaacattg 102300 atgactgtgc ccggggtccc cattgcctta atggtggtcagtgcatggat aggattggag 102360 gctacagttg tcgctgcttg cctggctttg ctggggagcgttgtgaggga gacatcaacg 102420 agtgcctctc caacccctgc agctctgagg gcagcctggactgtatacag ctcaccaatg 102480 actacctgtg tgtttgccgt agtgccttta ctggtgagaagctctctagc ctcctggcca 102540 ctctgccttg gaggatggtt ttagtgaaca aattgggccctgtgagttgt cccagaccca 102600 agtgaagctt ttcttatata agaaaaacaa cagcagcttaattttttaaa gccagttttt 102660 tcttagtctg tactttatgg atgttatgat cagtgtctacctttaatttt tcctaagaga 102720 ttcttttgtt gtgcattgat tatatcatgc tatggcaggaatatttttta caatgtatat 102780 tgaaagtagt tgaacaaaat aacatttttt aatagtggcaaaatatccaa aacctaaaat 102840 ttactatctg aatcatttta aagtgtacag tgtacagttcagtggcattg tttagtacat 102900 tgacattgtt gttctactat cacccccatt cattcacagaactcttttca tctgaacaaa 102960 ataacatttt gttttaagga accttacgtc taactccggtttcttcaccc tgcctccttt 103020 gacatgtgat ggcttgaatt actgggtagt tcctgccaccatgatctttc cacccctagg 103080 actcagagaa gcttaggacc ctgcttagat acttaaagggggcaggtcca aattcaagtg 103140 tgtttaacca aagatcagtt gatgtgggta tgaaatgaaaagttgcagtg gtcacaagta 103200 atgccttgcc cagtaaaggc ttggccatgt tcctactctgttcccaagct tctttctttc 103260 atcacagggc ctgaagatga ccaagctctg cctcagaataggggtcttat cacaagaact 103320 tactagttac ttctggagag ggcagcatgg tgacttttgggaactaaaat agttgtttgt 103380 atccagatgt ggcaggatac tcagatttga aatttatcatgcatttcctg gggtctgtag 103440 ttgtatttct tatatggagg gtgtgatcag attccttaggagtcagaaaa taggctttaa 103500 tagtctgttt acaaacaatt catcaagctg aactctgctacatcctgagc ccatctccag 103560 ccatctttcc ttccaacttt aaagatatgc taccttacaatcttggaatt ttgtaaagaa 103620 tgggagggat tctgcaatat ggaagctccc tggatacactaacatcaaaa aagaaatagt 103680 ccattgtgaa cataatagct attgaggcaa tgtggaacagaataagtatg aataatctgt 103740 ctacagcctc gtattggcca aactgcactt tgaaagccaaagcatcaaaa ctggctcctg 103800 tttctttcat tgcccctgag gcttttttct ctggaaacacaagcatttgc aaagtaaggg 103860 ggaagcagga gaaggaagga atggagacca ctttgaagtagtatcccaca gccaggaagt 103920 agtgtttgtg cattttccac atttgtgaat gaaaaaaaaaaaatcccttg aatacattga 103980 gcacagttat tgtgggatgc tgttgcagag ataaacagcaagggactggt ggtccaccaa 104040 gctgtgtcca tgtggtggct gctcttggtt agcggctgctgctgtgattt gaagagacaa 104100 acatttatac ctcttgctgc ttgatctcgt tggccaaaaacattactatt tttatttcat 104160 ttttaaagct acctcttctt tcccaagtga ccaccggccaaatggggcct atctcctgct 104220 gttggccagg agtcccagct gtgccctgcg tctcctctgtggttccagtt gcctcgttct 104280 ttatcttgga gcagtggccc ccaggggcct tccgttttagaactattcct agaggaaact 104340 aactctagga aaattaagct gtgttttaag gaatacgagttaggttacag aaaggccaaa 104400 aagtgattga aatgaggtat cacagatgaa aattctctataccgcaaatc accatttaaa 104460 tgtattggcc tggtgtagcc tctcctatga agtagataacattaacaaaa tgcagtgaat 104520 agtttttcaa ctgttttttt gcaacttatg aaagctaaaatgacttcaaa aagctgaagt 104580 gtgatttctc tttttttgat gggatttaag tcacttttctcacccaccct gttgtgtgag 104640 ttggtactta ctgtgtaggc aagacagagt aagggactggcctgagctgg tgacaggcca 104700 atcatgttga gatgcagtga gtgttttctt tctatgctcagattaccaca ggcctagaac 104760 tgaactctct cacacttgct tcacctatgg ctagttcccatcccagcaaa gtttgttgag 104820 taacttgcag tgactacttc tgccttttaa aaccttttttattttttatt ttctgaggcc 104880 ggcactgtga aaccttcgtc gatgtgtgtc cccagatgccctgcctgaat ggagggactt 104940 gtgctgtggc cagtaacatg cctgatggtt tcatttgccgttgtcccccg gtaagtgccc 105000 tgttgctcat ctaagaaggg gcattgtctc ttccatacaatctgtgaaca gaactgagat 105060 taaacctggt tttaccaatc ttcgaaatgg gaagatttctcaattttagc tctttcagag 105120 gaaagtgctt gggtttttcc atttaaagct gtttacaagtgacaaaacaa aacaaaaaca 105180 aataaaagct gtttagacac tcccagaaac agtgagcttcccaatccatg actgtcagtc 105240 ttaagaaaaa ctaagggaaa accacaaaaa gtggtggagtgcccccttga aaatagggct 105300 gatgtgtggg gcttcttggc cccaggctgg agggtaatctcactgcccag attgggccag 105360 agttggtgga aatgctgctc ttacattctt tagcaggaaccatttggaag caagttgtct 105420 cttaaagtag agggaagctc aattttagct atttgctatacccattcttt tatcactgcc 105480 agaggttgaa accttcccaa aacaagagat ccctatgcctcgggagatac caaaaggaaa 105540 cctgtctagt aaccttggct tgggctgaac gggaagtacagaagggagtg agatgtggag 105600 gttttctaga aacctcaaac ttccaatatg actgttttcctaccaatgcc atttttattt 105660 cctcactgat ttccacaggg attttccggg gcaaggtgccagagcagctg tggacaagtg 105720 aaatgtagga agggggagca gtgtgtgcac accgcctctggaccccgctg cttctgcccc 105780 agtccccggg actgcgagtc aggctgtgcc agtagcccctgccagcacgg gggcagctgc 105840 caccctcagc gccagcctcc ttattactcc tgccagtgtgccccaccatt ctcgggtagc 105900 cgctgtgaac tctacacggc accccccagc acccctcctgccacctgtct gagccagtat 105960 tgtgccgaca aagctcggga tggcgtctgt gatgaggcctgcaacagcca tgcctgccag 106020 tgggatgggg gtgactgttc tctcaccatg gagaacccctgggccaactg ctcctcccca 106080 cttccctgct gggattatat caacaaccag tgtgatgagctgtgcaacac ggtcgagtgc 106140 ctgtttgaca actttgaatg ccaggggaac agcaagacatgcaagtaagg gcttcacgtt 106200 ttccagaact gaaggggact gtcactaaca cacttcagtttctcgttctt gctcctaacc 106260 actccagtgc ttcagcctaa gctgcctctg cttttcagatggtgccagcc ttgtcccatc 106320 gcacactgcc ctgtgctctc ccttgctgac aatgggccaggaatgctcat tgcataatga 106380 ccctctctct ctgcctatct ttgccttctc acctgctgccttcgtgacct cctgggggat 106440 ataacttttg gaagtggtgt ttcccacttc aagcagcctctcacataacc tgcctgcagg 106500 atggatggaa aggtctgtat ctctctcttc accccattcctttccctaag actgtttttc 106560 cccatctaga tttatcagga agcatgaatt agattaagtctgtgaaccct cccggtctta 106620 attgtatagg atgctctatg gtttacttag gaactttcctattaactagc cattccatat 106680 aatttaatcc caattaattc tcagcaactc acgaggcataggtattacta acattctttc 106740 ataggtgaga gaacttaggc ccaagggtta aagaacttgccaaagcaagt tgtttctttg 106800 actcctaatc ttctgattga gctagcatga aaagttgtaccaaatagtat aaatatcaag 106860 gggtagggga tgaggagagg agagtataga tggaatagtttgtacatcat ttaggtttgg 106920 gggttctaat tccttttcca aatattgaaa gagcctgtacaagtaatatc tggtgtgcta 106980 taaggacata cccatctgcc tttaccttct gtagttgggattcactcttg aaagggaaag 107040 aaatgggtca tgttaatatt gttctctgta tggcaactctcaatggttca gggccttgta 107100 tcaagtttct agaggcatct ttcacttcct aaagcccgttgttaccttac tgggggcaat 107160 ggatttctct aaagtagagg aagcatgaac caggttgacttttgtttcag atatgcagct 107220 ttgtttgaaa acctgactta acaggcttgt aaactgcctgggagccccta agctcctgca 107280 gcatagtgag tacaatgttc ttcatttagg aagtactcaacaaggatgag tggcataggc 107340 atcagggata ggagatgcaa agaaggtttt gtggggcatttgggtcctgg gtgtgggcca 107400 cagaaaatgg gtaggaaaat ccacagtagt tccaaatgaaaaagcaaata gagctccagt 107460 ctaatctgag ctcttttgcc tgttctctgc ttccccttacctaggtatga caaatactgt 107520 gcagaccact tcaaagacaa ccactgtgac caggggtgcaacagtgagga gtgtggttgg 107580 gatgggctgg actgtgctgc tgaccaacct gagaacctggcagaaggtac cctggttatt 107640 gtggtattga tgccacctga acaactgctc caggatgctcgcagcttctt gcgggcactg 107700 ggtaccctgc tccacaccaa cctgcgcatt aagcgggactcccaggggga actcatggtg 107760 tacccctatt atggtgagaa gtcagctgct atgaagaaacagaggatgac acgcagatcc 107820 cttcctggtg aacaagaaca ggaggtggct gggtaggtgtttggtttctg aacttcaagg 107880 ctaattttat gaagcctagc actttgacat atagcacaagtcataactca acctatagaa 107940 atgcaaagat cagaaaaata gaatggccca ccacttaagataaacccgtt accccagtac 108000 ttacgtttaa atgtaaagat atgccttcgt cttggctgaaaagtgggcag gggtacacag 108060 gaggcagaat gtctggaagg cctcctcaga tgccagaatccataaccttt tgccagagtt 108120 gcctcaaatc cctttattag tgacattcct ttttacattaatcattgcct tattgttaag 108180 gcccttcttt agaatatatg tctctttgac acaggtaatgatcttcagtt gctctgtcat 108240 tcacacctca ctctttgttg gtatattgtc aaactttcaaccctccagct agctgagata 108300 ccaatgcaga gacaaaggaa aataaggact aaaaacacacagggatgtat gtttagagta 108360 gcaccaagta ccactgagta ggtatgaaag agtggaagaagaccactgaa accttcggga 108420 ggaaagcaca gcttctcact ttgtcatgca ttgcctcagccagaaggctt tggtcagcca 108480 gactaaggaa ggtgggacat cctctatgtg ggaattagtgtttaagatct caagggacag 108540 tattctaatt cctgagatat gtcttaaggc agcatcccagaggtggggac ctgatgctga 108600 aggggaggaa gggaggctcc tgtcaccctt tatgacatgttctgcctgac ctgcactctt 108660 ctgttttacc cccatctctc ctcctccgct tctcccttggcatcccacag ctctaaagtc 108720 tttctggaaa ttgacaaccg ccagtgtgtt caagactcagaccactgctt caagaacacg 108780 gatgcagcag cagctctcct ggcctctcac gccatacaggggaccctgtc ataccctctt 108840 gtgtctgtcg tcagtgagtg gcactggtgt aaaggggaaaggtggcgggg gaagagtgtc 108900 aattggggga acaattttca ttatgtagcc ctctattttttcaggactgt tgaggtcaaa 108960 gttgccctct aaatgaatct aatgctgaca ttgagaggttaatgttttat tcagcacttt 109020 ccctgtctct gtggactttc aggtgaatcc ctgactccagaacgcactca gctcctctat 109080 ctccttgctg ttgctgttgt catcattctg tttattattctgctgggggt aatcatggca 109140 aaacgaaagc gtaagcatgg ctctctctgg ctgcctgaaggtttcactct tcgccgagat 109200 gcaagcaatc acaagcgtcg tgagccagtg ggacaggatgctgtggggct gaagtaagag 109260 gcattgccaa taagccacta ttcacagtat aagtagggatgactagaaaa gcatatcttg 109320 ttgaatcatg attttaaatt ctaagctctg ggcatttactcacattgacc atgattctaa 109380 atgccttgtg agtgactgaa accacagtca gagcgtgggtgcatctattc tgtgtatatt 109440 atcacaatgc cgcccagctt ctgtgttgag gtgcccttttgttgtaagct cacgatcaat 109500 gggaagggaa aggatgaatg ccaagggaat gtcaccaaaaatactgccca tcttctctgg 109560 gccaagaccc tgcaaaattg taatatagcc acgggagtgtgtcatggtgg aaagtgttga 109620 aaaccactgt attagtttat gactgccatt cttattttttctttttataa tcatattaga 109680 aatctctcag tgcaagtctc agaagctaac ctaattggtactggaacaag tgaacactgg 109740 gtcgatgatg aagggcccca gccaaagaaa gtaaaggtgaggtggccatg gtagaaccac 109800 cattgtcagc cattatctgg ttacatttca aatttctgtcctttagctcc agagtgtcct 109860 gaatgggagt aaagagtgat agtcttgatg tcttacccagacaataacta tcctaggttc 109920 tcctaaacag caggaatcct taggacgtaa atgaccttgggttacctata tatgctcttc 109980 aagaataact ctttaaaaat actaagcaca tctgtttcctttagtgttat gctggcagta 110040 gtcaagtcag gaatggtgtg tgtagatggc aattccttcatggccacatc tcttccttat 110100 cctttatggg cacttcttct ataagagaag atgaaattaagcagtggaag aaagccagtt 110160 atcattgaca ttggcaaaat gaaagccagt gatcatggcaaaacgttgac attggaggct 110220 ccataccttg agcccaggaa actaaagcca tataaaagcaagtggtagat tgggctgaga 110280 ctgacatgaa aaagcagacc atccatccac agctgtgcatggctgggagc tggtggagca 110340 tgatggttaa gaagattgct ttagttaata tcagaactggattcaaatcc aaaactctgc 110400 cgcttagtag ctggcaagtt acttaacctc tttgtaattatccttccttg taaaatgcgg 110460 ataataataa ccctttcctc tgtgagttgt tgtgttggcatatgtgcctt atctactttc 110520 ctgtcatgta ctaggtattc agtaaagggt agctgcttttaaattatcat gattattatt 110580 attgaatagt ctttcctgag tgcttactac atgttgggtgctatgttgag tacaaggaaa 110640 caaccttgaa caaggcttat tttcccttct tgagaagcttacagccttgt ggtggagatg 110700 gccatgtagt gctgtgtaag catttaacac atgatcagttaagcagaggt aagcagagta 110760 tgctttttag ggccaacaag aagaattaaa gtttactttctagtggaggg taggaaaagt 110820 gagaattggg tgctctggag atctaaaggc taaagcctagagtttgggat tgggggaggg 110880 agttgtctag atgatgcagg aaaggtaagc aggagccacatcacgagagc agtgggaagc 110940 caaggctgag gtaatcctct ggaggggtgg aggagtaggaagagagaaaa caatgggaga 111000 taagcagcgg tggtggaggc tctggacacc ttctgattagcagtttctgg tggtggccag 111060 attgataggg agcattgttt tcacctttca ggctgaagatgaggccttac tctcagaaga 111120 agatgacccc attgatcgac ggccatggac acagcagcaccttgaagctg cagacatccg 111180 taggacacca tcgctggctc tcacccctcc tcaggcagagcaggaggtgg atgtgttaga 111240 tgtgaatgtc cgtggcccag gtcagcaccg ggcctcttcccctcttcact gggctgtgtc 111300 tgcccatggt tccacattgt tgtccctgtg tttgccctgtccagtcataa accccttccc 111360 aatttcacag actctaaatc agctcgagga aatgaatgcttaaggaatgt agccacaaag 111420 aaattatcag ctcagaaagg gaaggatttc acattgcttcatgtacagtg tgttgagact 111480 aaacaggctg ataaaatggg ttagattgta gtgggatagaaaaataatat cccaaagcag 111540 aggatagtta gtatcttatg catgtattaa ttgttctattaatattagat atttttaaat 111600 gtccaattaa aataacagca cttaaacttg aacagaaaaacgtgtttaga gaagagcccc 111660 cagcttaaaa agacttgtac tttcccatta ttctttacagaaatgatact atcagaggaa 111720 aaggtggcaa atgtgaaact aataaccagt tactacaagaagtacaggga attctttgcc 111780 cctaagaata actttagtca aatagagctg tttcaaccatagggttaact accttctcct 111840 tcttgagtac tttatggaat tttccattcc tacagatggctgcaccccat tgatgttggc 111900 ttctctccga ggaggcagct cagatttgag tgatgaagatgaagatgcag aggactcttc 111960 tgctaacatc atcacagact tggtctacca gggtgccagcctccaggccc agacagaccg 112020 gactggtgag atggccctgc accttgcagc ccgctactcacgggctgatg ctgccaagcg 112080 tctcctggat gcaggtgcag atgccaatgc ccaggacaacatgggccgct gtccactcca 112140 tgctgcagtg gcagctgatg cccaaggtgt cttccaggtaaagaacatga gaagagtcac 112200 ttgaataata attcctatgg ctctataatg ttttatcgtttttaaagaat taatatctca 112260 ttgaatttat tcaatcattt gagagattag ggcaggtattagagtcatgc ttggtagatg 112320 ggaaagtggg aaaagggagg caggcaaatt gacctagctatatataggaa gatagtgata 112380 gagccaactg cagacaacac cagtctttct gattattagtgataatttgt tgtgccaaga 112440 tgcctcccaa atgcgtagat tagatgagag tgatagctggtccggagcct ttgactagta 112500 tccgctactt cttatggatt ggcctgactg tatgggcttctagctgctca ttggcagttt 112560 tcttgacctg agttcatatt atattccctg atgcctgtttatccttatga ggaaattgga 112620 agcattctga atcaccttag gctgtatcct gactgaagttgtgatatttc cccacatccc 112680 tcttctttcc tgagaaagta gaaggctcat cactttacatcagccattat ttattgagca 112740 tgtatttatt tattcatgca acaaatattt attgattgatcatgatagaa tcttgggcac 112800 aaagataaca agactttccc tctcctccag gaatctacactgtagcctca gctctgatgc 112860 ccttgtttct tcactgagtt tcattatttt ccttcatccaggtgactctc ttttctgtac 112920 tttctcctct tctagattct gattcgcaac cgagtaactgatctagatgc caggatgaat 112980 gatggtacta cacccctgat cctggctgcc cgcctggctgtggagggaat ggtggcagaa 113040 ctgatcaact gccaagcgga tgtgaatgca gtggatgaccatggtaggga tgaaaggcag 113100 ggatttggat tcttacctgc aaagcatgcc tctgacgaggggaatagagt tacgtgaccc 113160 ttagagaact gcctgagatc caaatacgtg agagattcactcattctttc attcatgcat 113220 ttatttaaca tgctgagctc agagatccaa gccagtaggagatccctaat taaaatgagg 113280 gatttttaat gagggttctt ggacctcaga gcagatacatgaaaggtgtt cccatgtagt 113340 ttcctaggca taacacttga tttgaggatt tatgagagctgacttctcta ggagagttgt 113400 agaatattct caatataaat tctgacatgt aatatataggtttttatctt cagtatgtaa 113460 cagggtagat tctgccatgt aggggacatg aatgcctcatacaagtgatc ctttcaccta 113520 gaggtcttct gcaggctagg cagcctgtac tgaagtatttctagaacaga ggaaaaagaa 113580 gagattactt tggtttttcc tatagacccc tacagagaactgcttcctgc tgatctctgg 113640 ggagtttaag atttcattgt gcaagtcata gtgtcttatactaagagtag gatatgtctg 113700 tttataaaaa tgtttctccc ctgcaggaaa atctgctcttcactgggcag ctgctgtcaa 113760 taatgtggag gcaactcttt tgttgttgaa aaatggggccaaccgagaca tgcaggacaa 113820 caaggtacag tttgtgggct ctgagcttga ggaggctcatagcagtgcct cagttataag 113880 cccattcccg tttctcctta cagcttctct tgttatatgtatagacaaag atggaagcag 113940 tttgggcaga cactgggttt cgagagcttg atgaggatattgggcttgtc caggcatgtg 114000 tcaggatgga gttctctgcc ctctcagtac tcagcttgcagtaggctggc tatagatagc 114060 ttggtgtaac tctttctctt ccctgacatt tccttttctccagttgtctc ttctgttcat 114120 aatcacttct ttttctcgtt ttctttcttt tttccctccggctgcttttt tttctgtcct 114180 tggataccct gatgttttcc tgtttgattt ttctccatctgagttttttg tcctgcttcc 114240 ttctctcatt catcttctct cagttttctt tgttttccttatcttagtcc tttcttgcac 114300 atgttttgtc tcttctttca ctgtttccct tacttttcccagttctgtgc atgccttagt 114360 tttttctctt cctacctttc ttatttctaa ctccttttcctattatttct tatttctaac 114420 tcctattcct acctttctta tttctaactc cttttccttaaccttatttt gataagctca 114480 gtactctttt aagtgagtct ctggctcagg ggcatgctttattttatgaa ttcccaacta 114540 aagagatgtt ccccctaagg ggcaaaattg tttttttgaggagaggtgaa ataatcttac 114600 tttttttatg tgtaaatcac agatacacat tcagtacattggtagaggca cagcctatct 114660 gtggtgttaa tatttcatcg gggagataat taggaaaacatgtctaaggg ggtgataata 114720 aaaaagattg agaaaaactg ctatattctc aagagtgttattaacatgtg ttctgtgatg 114780 ggccttttct gtaggaagag acacctctgt ttcttgctgcccgggagggg agctatgaag 114840 cagccaagat cctgttagac cattttgcca atcgagacatcacagaccat atggatcgtc 114900 ttccccggga tgtggctcgg gatcgcatgc accatgacattgtgcgcctt ctggatgaat 114960 acaatgtgac cccaagccct ccaggcaccg tgttgacttctgctctctca cctgtcatct 115020 gtgggcccaa cagatctttc ctcagcctga agcacaccccaatgggcaag aagtctagac 115080 ggcccagtgc caagagtacc atgcctacta gcctccctaaccttgccaag gaggcaaagg 115140 atgccaaggg tagtaggagg aagaagtctc tgagtgagaaggtccaactg tctgagagtt 115200 cagtaacttt atcccctgtt gattccctag aatctcctcacacgtatgtt tccgacacca 115260 catcctctcc aatgattaca tcccctggga tcttacaggcctcacccaac cctatgttgg 115320 ccactgccgc ccctcctgcc ccagtccatg cccagcatgcactatctttt tctaaccttc 115380 atgaaatgca gcctttggca catggggcca gcactgtgcttccctcagtg agccagttgc 115440 tatcccacca ccacattgtg tctccaggca gtggcagtgctggaagcttg agtaggctcc 115500 atccagtccc agtcccagca gattggatga accgcatggaggtgaatgag acccagtaca 115560 atgagatgtt tggtatggtc ctggctccag ctgagggcacccatcctggc atagctcccc 115620 agagcaggcc acctgaaggg aagcacataa ccacccctcgggagcccttg ccccccattg 115680 tgactttcca gctcatccct aaaggcagta ttgcccaaccagcgggggct ccccagcctc 115740 agtccacctg ccctccagct gttgcgggcc ccctgcccaccatgtaccag attccagaaa 115800 tggcccgttt gcccagtgtg gctttcccca ctgccatgatgccccagcag gacgggcagg 115860 tagctcagac cattctccca gcctatcatc ctttcccagcctctgtgggc aagtacccca 115920 cacccccttc acagcacagt tatgcttcct caaatgctgctgagcgaaca cccagtcaca 115980 gtggtcacct ccagggtgag catccctacc tgacaccatccccagagtct cctgaccagt 116040 ggtcaagttc atcaccccac tctgcttctg actggtcagatgtgaccacc agccctaccc 116100 ctgggggtgc tggaggaggt cagcggggac ctgggacacacatgtctgag ccaccacaca 116160 acaacatgca ggtttatgcg tgagagagtc cacctccagtgtagagacat aactgacttt 116220 tgtaaatgct gctgaggaac aaatgaaggt catccgggagagaaatgaag aaatctctgg 116280 agccagcttc tagaggtagg aaagagaaga tgttcttattcagataatgc aagagaagca 116340 attcgtcagt ttcactgggt atctgcaagg cttattgattattctaatct aataagacaa 116400 gtttgtggaa atgcaagatg aatacaagcc ttgggtccatgtttactctc ttctatttgg 116460 agaataagat ggatgcttat tgaagcccag acattcttgcagcttggact gcattttaag 116520 ccctgcaggc ttctgccata tccatgagaa gattctacactagcgtcctg ttgggaatta 116580 tgccctggaa ttctgcctga attgacctac gcatctcctcctccttggac attcttttgt 116640 cttcatttgg tgcttttggt tttgcacctc tccgtgattgtagccctacc agcatgttat 116700 agggcaagac ctttgtgctt ttgatcattc tggcccatgaaagcaacttt ggtctccttt 116760 cccctcctgt cttcccggta tcccttggag tctcacaaggtttactttgg tatggttctc 116820 agcacaaacc tttcaagtat gttgtttctt tggaaaatggacatactgta ttgtgttctc 116880 ctgcatatat cattcctgga gagagaaggg gagaagaatacttttcttca acaaattttg 116940 ggggcaggag atcccttcaa gaggctgcac cttaatttttcttgtctgtg tgcaggtctt 117000 catataaact ttaccaggaa gaagggtgtg agtttgttgtttttctgtgt atgggcctgg 117060 tcagtgtaaa gttttatcct tgatagtcta gttactatgaccctccccac ttttttaaaa 117120 ccagaaaaag gtttggaatg ttggaatgac caagagacaagttaactcgt gcaagagcca 117180 gttacccacc cacaggtccc cctacttcct gccaagcattccattgactg cctgtatgga 117240 acacatttgt cccagatctg agcattctag gcctgtttcactcactcacc cagcatatga 117300 aactagtctt aactgttgag cctttccttt catatccacagaagacactg tctcaaatgt 117360 tgtacccttg ccatttagga ctgaactttc cttagcccaagggacccagt gacagttgtc 117420 ttccgtttgt cagatgatca gtctctactg attatcttgctgcttaaagg cctgctcacc 117480 aatctttctt tcacaccgtg tggtccgtgt tactggtatacccagtatgt tctcactgaa 117540 gacatggact ttatatgttc aagtgcagga attggaaagttggacttgtt ttctatgatc 117600 caaaacagcc ctataagaag gttggaaaag gaggaactatatagcagcct ttgctatttt 117660 ctgctaccat ttcttttcct ctgaagcggc catgacattccctttggcaa ctaacgtaga 117720 aactcaacag aacattttcc tttcctagag tcaccttttagatgataatg gacaactata 117780 gacttgctca ttgttcagac tgattgcccc tcacctgaatccactctctg tattcatgct 117840 cttggcaatt tctttgactt tcttttaagg gcagaagcattttagttaat tgtagataaa 117900 gaatagtttt cttcctcttc tccttgggcc agttaataattggtccatgg ctacactgca 117960 acttccgtcc agtgctgtga tgcccatgac acctgcaaaataagttctgc ctgggcattt 118020 tgtagatatt aacaggtgaa ttcccgactc ttttggtttgaatgacagtt ctcattcctt 118080 ctatggctgc aagtatgcat cagtgcttcc cacttacctgatttgtctgt cggtggcccc 118140 atatggaaac cctgcgtgtc tgttggcata atagtttacaaatggttttt tcagtcctat 118200 ccaaatttat tgaaccaaca aaaataatta cttctgccctgagataagca gattaagttt 118260 gttcattctc tgctttattc tctccatgtg gcaacattctgtcagcctct ttcatagtgt 118320 gcaaacattt tatcattcta aatggtgact ctctgcccttggacccattt attattcaca 118380 gatggggaga acctatctgc atggacctct gtggaccacagcgtacctgc ccctttctgc 118440 cctcctgctc cagccccact tctgaaagta tcagctactgatccagccac tggatatttt 118500 atatcctccc ttttccttaa gcacaatgtc agaccaaattgcttgtttct ttttcttgga 118560 ctactttaat ttggatcctt tgggtttgga gaaagggaatgtgaaagctg tcattacaga 118620 caacaggttt cagtgatgag gaggacaaca ctgcctttcaaactttttac tgatctctta 118680 gattttaaga actcttgaat tgtgtggtat ctaataaaagggaaggtaag atggataatc 118740 actttctcat ttgggttctg aattggagac tcagtttttatgagacacat cttttatgcc 118800 atgtatagat cctcccctgc tatttttggt ttatttttattgttataaat gctttctttc 118860 tttgactcct cttctgcctg cctttgggga taggtttttttgtttgttta tttgcttcct 118920 ctgttttgtt ttaagcatca ttttcttatg tgaggtggggaagggaaagg tatgagggaa 118980 agagagtctg agaattaaaa tattttagta taagcaattggctgtgatgc tcaaatccat 119040 tgcatcctct tattgaattt gccaatttgt aatttttgcataataaagaa ccaaaggtgt 119100 aatgttttgt tgagaggtgg tttagggatt ttggccctaaccaatacatt gaatgtatga 119160 tgactatttg ggaggacaca tttatgtacc cagaggcccccactaataag tggtactatg 119220 gttacttcct tgtgtacatt tctcttaaaa gtgatattatatctgtttgt atgagaaacc 119280 cagtaaccaa taaaatgacc gcatattcct gactaaacgtagtaaggaaa atgcacactt 119340 tgtttttact tttccgtttc attctaaagg tagttaagatgaaatttata tgaaagcatt 119400 tttatcacaa aataaaaaag gtttgccaag ctcagtggtgttgtattttt tattttccaa 119460 tactgcatcc atggcctggc agtgttacct catgatgtcataatttgctg agagagcaaa 119520 ttttcttttc tttctgaatc ccacaaagcc tagcaccaaacttctttttt tcttccttta 119580 attagatcat aaataaatga tcctggggaa aaagcatctgtcaaatagga aacatcacaa 119640 aactgagcac tcttctgtgc actagccata gctggtgacaaacagatggt tgctcaggga 119700 caaggtgcct tccaatggaa atgcgaagta gttgctatagcaagaattgg gaactgggat 119760 ataagtcata atattaatta tgctgttatg taaatgattggtttgtaaca ttccttaagt 119820 gaaatttgtg tagaacttaa tatacaggat tataaaataatattttgtgt ataaatttgt 119880 tataagttca cattcataca tttatttata aagtcagtgagatatttgaa catgaatact 119940 tgatgttgtt aatatgtatg gcattagcag tctcatattgaccctagcat cttatttcca 120000 ttttccatta ttttgcagaa acttatcagt cacacatgcatagttataaa tggtaacagt 120060 ttttttttta atgacttgaa atcttaggat atgcttctgctaagcagata aaggagcctt 120120 gttcaagatc tgcacagtta aggttttaac gtaatttagcattaattggt tatgttcaga 120180 atgttttaat tttatataaa acattttagt cttcatattgggttttataa tggtggaaga 120240 ataaaatgaa tttgagagct aaggaatgat cacctgtgcaggccggccac tcagagctca 120300 agttgccact tgcaaactat ttgggaacta aattgctccatgaaaacctg cttcagtgga 120360 tccaggttgc aggccattgg gtggctgacc acatcctgcccctgcaattt taagtcttga 120420 atgctagcct ttgattatgc agggaggact cggggcactcccagacgtga aagccaaaaa 120480 aggagagtag ggccgaagcc aagtgccacc ccatcacaaagctggagcag taggcttgga 120540 cactagacca tgagcatgcc aggagagggg ctagtgcctcagtcatgcaa ctaaaaagct 120600 tcacgaccca aggagagggg ctgcttcctt tatcagaacaacctccttaa tgcccacatg 120660 tttttcagga tgtgggcagt gggggaactg cgagcctgttaattgcctct gccactacgt 120720 ctttcagcca cagggtatat gaagttgtgt gtgtgttgtgtgtgtgaaag aatacaactt 120780 ttccttcttt tgccttagca cttagcaaaa cacatttaaaatatttgtac cagtcaagtc 120840 ccatccacaa agtgtgggca cactcaaatt aggatacatcaaagagggct atttacaaag 120900 gattaattac aaaagtgtgt gtgcaatgtc agggaccgacacacagttgt gcagaaaccc 120960 gtgttagcat cagcagagct atttccactt gtgggcccaaaaggccagga gaagtcacta 121020 ggacctggaa gcagagtcct gtagagcagg tccccaaccccccgggccac agactggata 121080 ccttagcctg tggggaaccg ttaggaaccg ggctgcacagcaggaggtga gcagcaggca 121140 agcaagcatt actgcctgag ctccacctcc tgtcagatcagcagtggcgt tagattctca 121200 taggagcatg aaccctattg tgaactgcat atgtgaaggatctaggttgc gcactcctta 121260 tgataatcta atgtctgatg atctgagatg gaagagtttcatccccaaac cattccctgg 121320 ccccagtcca tggaaatatt gtcttccacg aaactggttcctggtgccaa aaaggttgga 121380 gactgctgct gtagagcagg ctacctcaag aagagctgtgacttccttca aagc 121434 12 1161 DNA H. sapiens 12 ggcacgaggg ctgcacacccgagaaagttt cagccaaact tcgggcggcg gctgaggcgg 60 cggccgagga gcggcggactcggggcgcgg ggagtcgagg catttgcgct gggcttcgga 120 gcgtagcgcc agggcctgagcctttgaagc aggaggaggg gaggagagag tggggctcct 180 ctatcgggac cccctccccatgtggatctg cccaggcggc ggcggcggcg gcggcggagg 240 aggcgaccga gaagatacccgccctgcgcc ccgctctgct gtgggcgctg ctggcgctct 300 ggctgtgctg cgcgacccccgcgcatgcat tgcagtgtcg agatggctat gaaccctgtg 360 taaatgaagg aatgtgtgttacctaccaca atggcacagg atactgcaaa tgtccagaag 420 gcttcttggg ggaatattgtcaacatcgag acccctgtga gaagaaccgc tgccagaatg 480 gtgggacttg tgtggcccaggccatgctgg ggaaagccac gtgccgatgt gcctcagggt 540 ttacaggaga ggactgccagtactcgacat ctcatccatg ctttgtgtct cgaccttgcc 600 tgaatggcgg cacatgccatatgctcagcc gggataccta tgagtgcacc tgtcaagtcg 660 ggtttacagg taaggagtgccaatggaccg atgcctgcct gtctcatccc tgtgcaaatg 720 gaagtacctg taccactgtggccaaccagt tctcctgcaa atgcctcaca ggcttcacag 780 ggcagaagtg tgagactgatgtcaatgagt gtgacattcc aggacactgc cagcatggtg 840 gcacctgcct caacctgcctggttcctacc agtgccagtg ccttcagggc ttcacaggcc 900 agtactgtga cagcctgtatgtgccctgtg caccctcgcc ttgtgtcaat ggaggcacct 960 gtcggcagac tggtgacttcacttttgagt gcaactgcct tccagaaaca gtgagaagag 1020 gaacagagct ctgggaaagagacagggaag tctggaatgg aaaagaacac gatgagaatt 1080 agacactgga aaatatgtatgtgtggttaa taaagtgctt taaactgaat tgaaaaaaaa 1140 aaaaaaaaaa aaaaaaaaaa a1161 13 598 DNA H. sapiens 13 agcggggcgc ttcttcctgc gggaaacccctgggtgccca aggcggcggg gccgaggccg 60 cggcgacagt ggggcggggc ttgcggtgggaggaggcggc tgaggcggaa ggacacacga 120 ggctgcttcg ctgcacaccc gagaaagtttcagccaaact tcgggcggcg gctgaggcgg 180 cggccgagga gcggcggact cggggcgcggggagtcgagg catttgcgct gggcttcgga 240 gcgtagcgcc aggcctgagc ctttgaagcaggaggagggg aggagagagt ggggctcctc 300 tatcgggacc ccctccccat gtggatctggcccaggcggc ggcggcggcg gcggcggagg 360 aggcgaccga gaagataccc gccctgcgccccgctctgct gtgggcgctg ctggcgctct 420 ggctgtgctg cgcgaccccg cgcatgcattgcagtgtcga gatggctatg aaccctgtgt 480 acatgaagga ctgtgtgtta cctaccacaatggcacagga tactgcaaat gtccagaagg 540 cttctggggg gaatatgtca acatcgagacccctgttgag aagaaccgtg ccagaatg 598 14 20 DNA Artificial SequenceAntisense Oligonucleotide 14 tctcgatgtt gacaatattc 20 15 20 DNAArtificial Sequence Antisense Oligonucleotide 15 cactccttac ctgtaaaccc20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 ggcatttgcaggagaactgg 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17gtgctccctt caaaacctgg 20 18 20 DNA Artificial Sequence AntisenseOligonucleotide 18 tttcctgcat gctcacaagg 20 19 20 DNA ArtificialSequence Antisense Oligonucleotide 19 ccttcagaca ctcacagtgg 20 20 20 DNAArtificial Sequence Antisense Oligonucleotide 20 ctttgaaacc tggcatgcac20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 caatgcacacctttgaaacc 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22tagccattcg ggtgatcgat 20 23 20 DNA Artificial Sequence AntisenseOligonucleotide 23 attcatagcc attcgggtga 20 24 20 DNA ArtificialSequence Antisense Oligonucleotide 24 ctggcattca tagccattcg 20 25 20 DNAArtificial Sequence Antisense Oligonucleotide 25 ggcacactgg cattcatagc20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 cctgtggcacactggcattc 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27ttgcagatgc aggtgtagga 20 28 20 DNA Artificial Sequence AntisenseOligonucleotide 28 cattcatcaa tctggtcact 20 29 20 DNA ArtificialSequence Antisense Oligonucleotide 29 gtcatcaaaa ttaatttcac 20 30 20 DNAArtificial Sequence Antisense Oligonucleotide 30 gcacagtcat caaaattaat20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 tggaggcacactcatcaatg 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32ttcacctgtg agtagcagct 20 33 20 DNA Artificial Sequence AntisenseOligonucleotide 33 agacctccag tacagtttcc 20 34 20 DNA ArtificialSequence Antisense Oligonucleotide 34 cactgagacc tccagtacag 20 35 20 DNAArtificial Sequence Antisense Oligonucleotide 35 aagcaggttc cttggttcag20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 tgccagccaggagcacacaa 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37tggcaaggat tggcaaggca 20 38 20 DNA Artificial Sequence AntisenseOligonucleotide 38 ttcatgtctg tctggcactt 20 39 20 DNA ArtificialSequence Antisense Oligonucleotide 39 agggtctgac agtttttccc 20 40 20 DNAArtificial Sequence Antisense Oligonucleotide 40 tcaccagggt ctgacagttt20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 gctggcacaagtgttcaaca 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42tcacagttga caccctgata 20 43 20 DNA Artificial Sequence AntisenseOligonucleotide 43 catactcaca gttgacaccc 20 44 20 DNA ArtificialSequence Antisense Oligonucleotide 44 cacttcatac tcacagttga 20 45 20 DNAArtificial Sequence Antisense Oligonucleotide 45 tccccagcaa agccaggcaa20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 acttgtccacagctgctctg 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47ccatcccact ggcaggcatg 20 48 20 DNA Artificial Sequence AntisenseOligonucleotide 48 tcatcacact ggttgttgat 20 49 20 DNA ArtificialSequence Antisense Oligonucleotide 49 aagttgtcaa acaggcactc 20 50 20 DNAArtificial Sequence Antisense Oligonucleotide 50 cagtatttgt catacttgca20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 ctgcacagtatttgtcatac 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52gtggtctgca cagtatttgt 20 53 20 DNA Artificial Sequence AntisenseOligonucleotide 53 ttgaagtggt ctgcacagta 20 54 20 DNA ArtificialSequence Antisense Oligonucleotide 54 tgtctttgaa gtggtctgca 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 tcacagtggt tgtctttgaa20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gactttagagccagccacct 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57agcttcaagg tgctgctgtg 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 tctgcagctt caaggtgctg 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 gcctcctcgg agagaagcca 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 gagctgcctc ctcggagaga20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 accaagtctgtgatgatgtt 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62ggtagaccaa gtctgtgatg 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 gcaaggtgca gggccatctc 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 tttccatggt catccactgc 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 cagattttcc atggtcatcc20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 acattattgacagcagctgc 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67gcctccttgg caaggttagg 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 aagccacact gggcaaacgg 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 atggtctgag ctacctgccc 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 cccacagagg ctgggaaagg20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 acttgcccacagaggctggg 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72gtcacatctg accagtcaga 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 tggtggtcac atctgaccag 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 ggactctctc acgcataaac 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 aaaggtcttg ccctataaca20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 cattccaaacctttttctgg 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77cagacaaatc aggtaagtgg 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 caaaacatta cacctttggt 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 atttcactta aggaatgtta 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 tcaggagatc gagaccatcc20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 agctccttacctggaaggca 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82ccaaccacta cgggtcttgg 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 ccagtgaaac ctttggaaag 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 atgttcttac cttgccagcc 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 ggcatactca ctggcaaggc20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 aagcccttacttgcatgtct 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87gaatgacaga gcaactgaag 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 ctttctcggg tgtgcagccc 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 gtatcttctc ggtcgcctcc 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 cctgtctctt tcccagagct20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 cgccgccttgggcacccagg 20 92 20 DNA H. sapiens 92 gaatattgtc aacatcgaga 20 93 20 DNAH. sapiens 93 ccagttctcc tgcaaatgcc 20 94 20 DNA H. sapiens 94ccaggttttg aagggagcac 20 95 20 DNA H. sapiens 95 ccttgtgagc atgcaggaaa20 96 20 DNA H. sapiens 96 ccactgtgag tgtctgaagg 20 97 20 DNA H. sapiens97 gtgcatgcca ggtttcaaag 20 98 20 DNA H. sapiens 98 atcgatcacccgaatggcta 20 99 20 DNA H. sapiens 99 cgaatggcta tgaatgccag 20 100 20DNA H. sapiens 100 gaatgccagt gtgccacagg 20 101 20 DNA H. sapiens 101tcctacacct gcatctgcaa 20 102 20 DNA H. sapiens 102 gtgaaattaa ttttgatgac20 103 20 DNA H. sapiens 103 attaattttg atgactgtgc 20 104 20 DNA H.sapiens 104 cattgatgag tgtgcctcca 20 105 20 DNA H. sapiens 105agctgctact cacaggtgaa 20 106 20 DNA H. sapiens 106 ggaaactgta ctggaggtct20 107 20 DNA H. sapiens 107 ctgtactgga ggtctcagtg 20 108 20 DNA H.sapiens 108 ctgaaccaag gaacctgctt 20 109 20 DNA H. sapiens 109ttgtgtgctc ctggctggca 20 110 20 DNA H. sapiens 110 tgccttgcca atccttgcca20 111 20 DNA H. sapiens 111 aagtgccaga cagacatgaa 20 112 20 DNA H.sapiens 112 gggaaaaact gtcagaccct 20 113 20 DNA H. sapiens 113aaactgtcag accctggtga 20 114 20 DNA H. sapiens 114 tgttgaacac ttgtgccagc20 115 20 DNA H. sapiens 115 tatcagggtg tcaactgtga 20 116 20 DNA H.sapiens 116 tcaactgtga gtatgaagtg 20 117 20 DNA H. sapiens 117ttgcctggct ttgctgggga 20 118 20 DNA H. sapiens 118 cagagcagct gtggacaagt20 119 20 DNA H. sapiens 119 catgcctgcc agtgggatgg 20 120 20 DNA H.sapiens 120 atcaacaacc agtgtgatga 20 121 20 DNA H. sapiens 121gagtgcctgt ttgacaactt 20 122 20 DNA H. sapiens 122 tgcaagtatg acaaatactg20 123 20 DNA H. sapiens 123 gtatgacaaa tactgtgcag 20 124 20 DNA H.sapiens 124 acaaatactg tgcagaccac 20 125 20 DNA H. sapiens 125tactgtgcag accacttcaa 20 126 20 DNA H. sapiens 126 tgcagaccac ttcaaagaca20 127 20 DNA H. sapiens 127 ttcaaagaca accactgtga 20 128 20 DNA H.sapiens 128 cacagcagca ccttgaagct 20 129 20 DNA H. sapiens 129cagcaccttg aagctgcaga 20 130 20 DNA H. sapiens 130 tggcttctct ccgaggaggc20 131 20 DNA H. sapiens 131 aacatcatca cagacttggt 20 132 20 DNA H.sapiens 132 gcagtggatg accatggaaa 20 133 20 DNA H. sapiens 133ggatgaccat ggaaaatctg 20 134 20 DNA H. sapiens 134 gcagctgctg tcaataatgt20 135 20 DNA H. sapiens 135 cctaaccttg ccaaggaggc 20 136 20 DNA H.sapiens 136 ccgtttgccc agtgtggctt 20 137 20 DNA H. sapiens 137gggcaggtag ctcagaccat 20 138 20 DNA H. sapiens 138 cctttcccag cctctgtggg20 139 20 DNA H. sapiens 139 cccagcctct gtgggcaagt 20 140 20 DNA H.sapiens 140 tctgactggt cagatgtgac 20 141 20 DNA H. sapiens 141ctggtcagat gtgaccacca 20 142 20 DNA H. sapiens 142 gtttatgcgt gagagagtcc20 143 20 DNA H. sapiens 143 ccagaaaaag gtttggaatg 20 144 20 DNA H.sapiens 144 ccacttacct gatttgtctg 20 145 20 DNA H. sapiens 145accaaaggtg taatgttttg 20 146 20 DNA H. sapiens 146 tgccttccag gtaaggagct20 147 20 DNA H. sapiens 147 ccaagacccg tagtggttgg 20 148 20 DNA H.sapiens 148 ctttccaaag gtttcactgg 20 149 20 DNA H. sapiens 149gccttgccag tgagtatgcc 20 150 20 DNA H. sapiens 150 agacatgcaa gtaagggctt20 151 20 DNA H. sapiens 151 cttcagttgc tctgtcattc 20 152 20 DNA H.sapiens 152 ggaggcgacc gagaagatac 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding Notch2, wherein said compoundspecifically hybridizes with said nucleic acid molecule encoding Notch2(SEQ ID NO: 4) and inhibits the expression of Notch2.
 2. The compound ofclaim 1 comprising 12 to 50 nucleobases in length.
 3. The compound ofclaim 2 comprising 15 to 30 nucleobases in length.
 4. The compound ofclaim 1 comprising an oligonucleotide.
 5. The compound of claim 4comprising an antisense oligonucleotide.
 6. The compound of claim 4comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprisingan RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimericoligonucleotide.
 9. The compound of claim 4 wherein at least a portionof said compound hybridizes with RNA to form an oligonucleotide-RNAduplex.
 10. The compound of claim 1 having at least 70% complementaritywith a nucleic acid molecule encoding Notch2 (SEQ ID NO: 4) saidcompound specifically hybridizing to and inhibiting the expression ofNotch2.
 11. The compound of claim 1 having at least 80% complementaritywith a nucleic acid molecule encoding Notch2 (SEQ ID NO: 4) saidcompound specifically hybridizing to and inhibiting the expression ofNotch2.
 12. The compound of claim 1 having at least 90% complementaritywith a nucleic acid molecule encoding Notch2 (SEQ ID NO: 4) saidcompound specifically hybridizing to and inhibiting the expression ofNotch2.
 13. The compound of claim 1 having at least 95% complementaritywith a nucleic acid molecule encoding Notch2 (SEQ ID NO: 4) saidcompound specifically hybridizing to and inhibiting the expression ofNotch2.
 14. The compound of claim 1 having at least one modifiedinternucleoside linkage, sugar moiety, or nucleobase.
 15. The compoundof claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. Thecompound of claim 1 having at least one phosphorothioate internucleosidelinkage.
 17. The compound of claim 1 having at least one5-methylcytosine.
 18. A method of inhibiting the expression of Notch2 incells or tissues comprising contacting said cells or tissues with thecompound of claim 1 so that expression of Notch2 is inhibited.
 19. Amethod of screening for a modulator of Notch2, the method comprising thesteps of: a. contacting a preferred target segment of a nucleic acidmolecule encoding Notch2 (SEQ ID NO: 4) with one or more candidatemodulators of Notch2, and b. identifying one or more modulators ofNotch2 expression which modulate the expression of Notch2.
 20. Themethod of claim 19 wherein the modulator of Notch2 expression comprisesan oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide,an RNA oligonucleotide, an RNA oligonucleotide having at least a portionof said RNA oligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. Adiagnostic method for identifying a disease state comprising identifyingthe presence of Notch2 in a sample using at least one of the primerscomprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22.A kit or assay device comprising the compound of claim
 1. 23. A methodof treating an animal having a disease or condition associated withNotch2 comprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of Notch2 is inhibited.
 24. The method of claim 23 whereinthe disease or condition is an autoimmune disorder.